Il-17f-specific capture agents, compositions, and methods of using and making

ABSTRACT

The present application provides stable peptide-based IL-17F capture agents and methods of use as detection agents. The application further provides methods of manufacturing IL-17F capture agents.

RELATED APPLICATIONS

This application is a continuation of pending U.S. application Ser. No.15/211,759, filed Jul. 15, 2016, which claims priority from, and benefitof, U.S. Provisional Patent Application No. 62/192,899, filed on Jul.15, 2015, U.S. Provisional Patent Application No. 62/277,430, filed onJan. 11, 2016, and U.S. Provisional Patent Application No. 62/309,756,filed on Mar. 17, 2016, each of which is incorporated herein byreference in its entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Oct. 8, 2020, as a text file named“INDI_27_1_CON_AMD_AFD_Sequence_Listing.txt,” created on May 21, 2020,and having a size of 14,789 bytes is hereby incorporated by referencepursuant to 37 C.F.R. § 1.52(e)(5).

BACKGROUND

Human interleukin-17 (IL-17A) is a pro-inflammatory cytokine secreted byactivated T-cells. IL-17A is a validated target for treatment of severeplaque psoriasis. There are six different homodimeric cytokines(IL-17A-F) and the heterodimer IL-17A/F in the IL-17 cytokine familyIL-17F is the closest homologue to IL-17A and is 50% identical insequence. IL-17A and IL-17F are secreted both as disulfide-linkedhomodimers (32-38 kDa) and as the IL-17A/F covalent heterodimer (40-45kDa). Like IL-17A, IL-17F activates immune and non-immune cells toinduce pro-inflammatory mediators. These mediators can induce neutrophilrecruitment at inflammatory sites, promote local tissue destruction,induce neovascularization in tumors, enhance osteoclastogenesis, andprotect from pathogen infection, resulting in disease development andhost protection.

IL-17A is a validated target for treatment of severe plaque psoriasis.The current approach is to block IL-17A interaction with its receptor atthe cell surface by neutralizing circulating IL-17A. This is doneprimarily through monoclonal antibodies and fragments thereof.

IL-17 cytokine family members mediate their effects through binding tothe IL-17 receptor family, of which there are five related members(IL-17RA-IL-17RE). Both IL-17A and IL-17F bind as homodimers orheterodimers to the heterodimeric receptor complex formed betweenIL-17RA and IL-17RC. While the activity of IL-17F appears to be relatedto that of IL-17A, the potency differs, consistent with differences inreceptor binding affinities.

Because of their roles in immunity and immune-mediated diseases, IL-17Aand IL-17F have become an area of focus in therapeutic drug development.The very low natural abundance of circulating IL-17A and IL-17A/F hasbeen a challenge for detecting these biomarkers by traditional sandwichimmunoassays. Highly sensitive detection of the circulating levels ofeach homodimer (IL17A, IL-17F), as well as the IL-17A/F heterodimer,would be informative for understanding the involvement of each cytokineover the course of disease and treatment.

SUMMARY

The present disclosure relates to chemically synthesized capture agents(called protein-catalyzed capture agents, or PCC Agents) that aredesigned to bind to detect interleukin 17F (IL-17F), methods for makingsaid capture agents using iterative in situ click chemistry, methods forusing said capture agents to detect IL-17F, and assays employing saidmethods.

In one aspect, provided herein is a stable, synthetic capture agent thatspecifically binds IL-17F, wherein the capture agent comprises one ormore designed anchor ligands. In certain embodiments, the capture agentcomprises two anchor ligands joined by a linker. In another aspect,provided herein is a composition comprising one or more syntheticcapture agents, as described herein, that specifically bind IL-17F.According to certain embodiments, the capture agent binds IL-17F with agreater affinity than IL-17A. According to certain embodiments, thecapture agent binds IL-17F with at least 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 100 or 1000 greater affinity than IL-17A.

In another aspect, provided herein is a stable, synthetic capture agentthat specifically binds IL-17F, wherein the capture agent comprises afirst ligand having affinity for a first epitope on IL-17F, a secondligand having affinity for a second epitope on IL-17F, and a linkercovalently connecting the first ligand to the second ligand.Particularly, the first ligand binds the first epitope (or a syntheticversion thereof) in isolation and the second ligand binds the secondepitope (or a synthetic version thereof) in isolation. In the captureagent, the first ligand and the second ligand cooperatively bind thefirst and second epitopes of IL-17F, respectively.

In another aspect, provided herein is a method for detecting IL-17F in abiological sample, comprising the step of treating the biological samplewith one or more capture agents described herein.

Anchor Ligand

In one embodiment of the capture agent, the capture agent comprises twoligands that specifically bind IL-17F at two distinct epitopes. Theseanchor ligands (sometimes referred to herein as simply “ligands”) canthen be bound to each other by a linker that provides increased affinityfor IL-17F. In certain embodiments, there is a first ligand and a secondligand that bind to a first epitope and a second epitope, respectively.

According to certain embodiments, the first epitope comprises the aminoacid sequence of FFQKPES (SEQ ID NO:1) or FFQKPESCPPVPGG (SEQ ID NO:2).In certain embodiments, the first epitope is between 5 and 20 aminoacids long. In other embodiments, the first epitope is between 7 and 13amino acids long. In other embodiments, the first epitope is at most, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acidslong.

According to certain embodiments, the first epitope comprises the aminoacid sequence of NENQRVS (SEQ ID NO:3) or GIINENQRVS (SEQ ID NO:4). Incertain embodiments, the first epitope is between 5 and 20 amino acidslong. In other embodiments, the first epitope is between 7 and 10 aminoacids long. In other embodiments, the first epitope is at most, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long.

According to certain embodiments, the first ligand comprises an aminoacid sequence selected from FYKTH (SEQ ID NO:5), FYKQH (SEQ ID NO:6),FYLTH (SEQ ID NO:7), FYLQH (SEQ ID NO:8), RRATS (SEQ ID NO:9) and RRAQS(SEQ ID NO:10). According to certain embodiments, the first ligandcomprises an amino acid sequence selected from RRATS (SEQ ID NO:9) andRRAQS (SEQ ID NO:10). In certain embodiments, the first ligand iscyclic. In certain embodiments, the first ligand comprises a1,4-substituted-1,2,3-triazole residue (Tz4) or a1,5-substituted-1,2,3-triazole residue (Tz5).

According to certain embodiments, the first ligand comprises an aminoacid sequence selected from KYGEV (SEQ ID NO:11), LYGEV (SEQ ID NO:12),VHKSG (SEQ ID NO:13), VHLSG (SEQ ID NO:14), QKHGP (SEQ ID NO:15), TKHGP(SEQ ID NO:16), QLHGP (SEQ ID NO:17), TLHGP (SEQ ID NO:18), YDLQR (SEQID NO:19), YDLTR (SEQ ID NO:20), YDKQR (SEQ ID NO:21), YDKTR (SEQ IDNO:22), KKGWP (SEQ ID NO:23), KLGWP (SEQ ID NO:24), LKGWP (SEQ IDNO:25), LLGWP (SEQ ID NO:26), RSYNL (SEQ ID NO:27), and RSYNK (SEQ IDNO:28). According to certain embodiments, the first ligand comprises anamino acid sequence selected from TKHGP (SEQ ID NO:16), QKHGP (SEQ IDNO:15), KKGWP (SEQ ID NO:23) and RSYNK (SEQ ID NO:28). In certainembodiments, the first ligand is cyclic. In certain embodiments, thefirst ligand comprises a 1,4-substituted-1,2,3-triazole residue (Tz4) ora 1,5-substituted-1,2,3-triazole residue (Tz5).

According to certain embodiments, the first ligand comprises thesequence RRATS (SEQ ID NO:9) and the second ligand comprises thesequence QKHGP (SEQ ID NO:15). In other embodiments, the first ligandcomprises the sequence RRATS (SEQ ID NO:9) and the second ligandcomprises the sequence RSYNK (SEQ ID NO:28). In other embodiments, thefirst and second ligands are cyclic and comprise a Tz4 residue.

Linker

According to certain embodiments, the capture agent further comprises alinker that binds both the first and second ligand. According to certainembodiments, the length of the linker corresponds to distance betweenthe first epitope and the second epitope. The length of the linker mustbe at least the distance between the first and second epitopes. Incertain embodiments, the linker is longer than the distance between thefirst and second epitopes. According to certain embodiments, the linkeris 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%longer than the distance between the first and second epitopes.

According to certain embodiments, the linker is ˜4.4 Å to ˜26.4 Å, ˜8.8Å to ˜26.4 Å or ˜7 Å to ˜15 Å in length. In certain embodiments, thelength of the linker is ˜15 Å.

In other embodiments, the linker comprises one or more repeat units ofethylene glycol. In some embodiments, the linker is PEG₁, PEG₂, PEG₃,PEG₄, or PEG₅. In other embodiments, the linker comprises a peptide. Inother embodiments, the linker comprises an amino acid. In a particularembodiment, the linker is glycine. In other embodiments, the linkercomprises an alkylene moiety, wherein the alkylene moiety is optionallysubstituted with one or more moieties provided herein.

Triazole Linkage

In one embodiment of the capture agent, the anchor ligand and secondaryligand are linked together via a 1,4-substituted-1,2,3-triazole residue(Tz4). In another embodiment, the secondary ligand and the tertiaryligand are linked together via a 1,4-substituted-1,2,3-triazole residue(Tz4). In yet another embodiment, the tertiary ligand and thequarternary ligand are linked together via a1,4-substituted-1,2,3-triazole residue (Tz4). In yet another embodiment,the anchor ligand and secondary ligand are linked together via a1,4-substituted-1,2,3-triazole residue, and the secondary ligand and thetertiary ligand are linked together via a 1,4-substituted-1,2,3-triazoleresidue. In yet another embodiment, the anchor ligand and secondaryligand are linked together via a 1,4-substituted-1,2,3-triazole residue,the secondary ligand and the tertiary ligand are linked together via a1,4-substituted-1,2,3-triazole residue and the tertiary ligand and thequarternary ligand are linked together via a1,4-substituted-1,2,3-triazole residue.

Capture Agents

According to certain embodiments, the capture agent has a structureselected from the following:

According to other embodiments, the capture agent has a structureselected from the following:

Properties

In certain embodiments, the IL-17F capture agents provided herein arestable across a wide range of temperatures, pH values, storage times,storage conditions, and reaction conditions, and in certain embodimentsthe capture agents are more stable than a comparable antibody orbiologic. In certain embodiments, the capture agents are stable instorage as a lyophilized powder. In certain embodiment, the captureagents are stable in storage at a temperature of about −80° C. to about60° C. In certain embodiments, the capture agents are stable at roomtemperature. In certain embodiments, the capture agents are stable inhuman serum for at least 24 hours. In certain embodiments, the captureagents are stable at a pH in the range of about 3 to about 12. Incertain embodiments, the capture agents are stable as a powder for twomonths at a temperature of about 60° C.

Detectable Labels

In some embodiments, the capture agent is labeled with a label selectedfrom the group consisting of biotin, copper-DOTA, biotin-PEG3,aminooxyacetate, ¹⁹FB, ¹⁸FB and FITC-PEG3. In other embodiments, thecapture agent is labeled with the detectable moiety consisting of ⁶⁴CuDOTA, ⁶⁸Ga DOTA, ¹⁸F, ⁶⁴Cu, ⁶⁸Ga, ⁸⁹Zr, ¹²⁴I, ⁸⁶Y, ^(94m)Tc, ^(110m)In,¹¹C and ⁷⁶Br. In other embodiments, the label is a fluorescent label. Ina particular embodiment, the detectable label is ¹⁸F.

Methods and Uses

As used herein, the terms “capture agent of the invention”, or “captureagents of the invention” refer to synthetic protein-catalyzed captureagents which bind IL-17F, as described herein.

Also provided is a method of detecting IL-17F in a subject, comprisingthe step of contacting a biological sample from the subject with one ormore capture agents of the invention. Also provided is the use of one ormore capture agents of the invention for the detection of IL-17F in asubject.

Also provided is a method of detecting IL-17F in a biological sampleusing an immunoassay, wherein the immunoassay utilizes a capture agentas described herein, and wherein said capture agent replaces an antibodyor its equivalent in the immunoassay. In certain embodiments, methodsare provided for identifying, detecting, quantifying, or separatingIL-17F in a biological sample using the capture agents as describedherein. In one embodiment of the method, the immunoassay is selectedfrom the group of Western blot, pull-down assay, dot blot, and ELISA.

Also provided is a method of detecting the presence of IL-17F in a humanor mammalian subject, the method comprising the steps of:

-   -   (a) administering to a biological sample from the subject one or        more capture agents of the invention, wherein each capture agent        is linked to a detectable moiety; and    -   (b) detecting the moiety linked to each capture agent in the        subject; wherein detection of the moiety indicates the presence        of IL-17F in the subject.

Also provided herein is a method of detecting IL-17F in a samplecomprising:

-   -   (a) exposing the sample to one or more capture agents of the        invention, wherein each capture agent is linked to a detectable        moiety;    -   (b) binding IL-17F in the biological sample to a capture agent;        and    -   (c) detecting the moiety linked to each capture agent on the        substrate;

wherein detection of the moiety on the substrate detects IL-17F in thesample.

Kits

Provided herein in certain embodiments are kits comprising one or morecapture agents of the invention. In certain embodiments, these kits maybe used for identifying, detecting, quantifying, and/or separatingIL-17F, and in certain embodiments the kits may be used in the diagnosisand/or staging of a condition associated with the presence of IL-17F. Incertain embodiments, a kit as provided herein comprises: (a) a substratecomprising an adsorbent thereon, wherein the adsorbent is suitable forbinding IL-17F, and (b) a washing solution or instructions for making awashing solution, wherein the combination of the adsorbent and thewashing solution allows detection of IL-17F. In other embodiments, thekits provided herein may be used in the treatment of a conditionassociated with the presence of IL-17F.

In certain embodiments, a kit may further comprise instructions forsuitable operational parameters in the form of a label or a separateinsert. For example, the kit may have standard instructions informing aconsumer/kit user how to wash the probe after a sample of plasma orother tissue sample is contacted on the probe.

In certain embodiments, a kit comprises (a) one or more capture agentsthat specifically bind IL-17F; and (b) a detection reagent. Such kitscan be prepared from the materials described herein.

The kits provided herein may optionally comprise a standard or controlinformation, and/or a control amount of material, so that the testsample can be compared with the control information standard and/orcontrol amount to determine if the test amount of IL-17F detected in asample is an amount consistent with a diagnosis of a particularcondition.

Synthesis of Capture Agents

Provided herein are methods for making (i.e., synthesizing)IL-17F-specific capture agents of the invention. In one embodiment, themethod comprises the steps of:

-   -   (a) selecting a first ligand that binds to a first epitope on        the target protein,    -   (b) selecting a second ligand that binds to a second epitope on        the target protein,    -   (c) selecting a linker that has a length that allows the linker        to bind both the first ligand and the second ligand when both        the first and the second ligands are specifically binding the        first and second epitopes, respectively, and    -   (d) binding the linker to the first and second ligands, thereby        producing the synthetic capture agent that specifically binds to        the target protein.

In certain embodiments the ligands are identified using the followingsteps:

-   -   (1) a pre-clear to eliminate non-specific binders,    -   (2) a product screen to identify hits resulting from        epitope-templated in situ click chemistry,    -   (3) a target screen against His-tagged IL-17F protein, and    -   (4) another target screen against His-tagged IL-17F protein in        2% (v/v) human serum to identify peptides whose binding to        IL-17F is unperturbed by serum proteins.

In certain embodiments, the first epitope and the second epitope are˜4.4 Å to ˜26.4 Å, ˜8.8 Å to ˜26.4 Å or ˜7 Å to ˜15 Å or ˜15 Å distantfrom each other. In some embodiments, the linker is longer than thedistance between the first and second epitope. Optionally, the linker is10-50%, 5-25% or 1-10% longer than the distance between the first andsecond epitope.

In certain embodiments, the capture agent has a binding affinity for thetarget protein greater than either of the ligands. In some embodiments,the capture agent has a binding affinity that is at least 50, 75 or 90%of the binding affinity of a full cooperative binder. In otherembodiments, the capture agent has a binding affinity that is at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% ofthe binding affinity of a full cooperative binder.

In certain embodiments, the target protein is a synthetic epitope,wherein the synthetic epitope comprises at least a 20 amino acidsequence of a full length protein, wherein at least one amino acid ofthe synthetic epitope comprises an azide or an acetylene group. In someembodiments, the synthetic epitope is at least 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 150, 200, 250 or 300 amino acid sequence of afull length protein. In some embodiments, at least two amino acids ofthe synthetic epitope comprise an azide or an acetylene group. In otherembodiments, at least 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of thesynthetic epitope comprise an azide or an acetylene group.

According to certain embodiments, the full length protein is a naturallyoccurring protein. According to other embodiments, the naturallyoccurring protein is IL-17.

According to certain embodiments, the capture agent binds the syntheticepitope and the full length protein with a binding affinity that is atleast 50% of the binding affinity of a full cooperative binder.According to certain embodiments, the capture agent binds the syntheticepitope and the full length protein with a binding affinity that is atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or100% of the binding affinity of a full cooperative binder.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C: Epitopes derived from the IL-17F protein. FIG. 1A.N-terminal sequence differences occur in the mature proteins thatdiscriminate IL-17F (SEQ ID NO:38) from IL-17A (SEQ ID NO:37). Thisregion of uniqueness corresponds to Arg-31 to Thr-79 in IL-17F. FIG. 1B.Sequence of the designed Epitope1 fragment containing a biotin-PEG₃assay handle and a strategically substituted azide click handle (C48Az4;Az4=L-azidolysine), (Biotin-PEG₃-FFQKPES (SEQ ID NO:1) [Az4]PPVPGGS)(SEQID NO:32). FIG. 1C. Sequence of the designed Epitope2 fragmentcontaining a biotin-PEG₃ assay handle and a strategically substitutedazide click handle (I62Az4), (Biotin-PEG₃-GI[Az4]NENQRVS) (SEQ IDNO:33).

FIGS. 2A-2D: In vitro characterization of macrocycles developed againstIL-17F Epitope1. FIG. 2A. Sandwich ELISAs for human IL-17F proteinagainst PEG₃-biotin-modified Cy(RRATS) (SEQ ID NO:9) and Cy(RRAQS) (SEQID NO:10) yield EC₅₀ values of 66 to 52 nM. A similarly assayedbiotinylated monoclonal antibody (TA319597, Origene) shows similarbinding affinity. FIG. 2B. Point ELISAs for human IL-17F and IL-17Aproteins against the two macrocyclic peptide ligands,PEG₃-biotin-modified Cy(RRATS) (SEQ ID NO:9) and Cy(RRAQS) (SEQ IDNO:10), demonstrate preferential binding to IL-17F. FIG. 2C. Massspectrum of Cy(RRATS)(SEQ ID NO:9)-PEG₃-biotin. FIG. 2D. Mass spectrumof Cy(RRAQS) (SEQ ID NO:10)-PEG₃-biotin.

FIGS. 3A-3B: Orientation of macrocycle binding to IL-17F Epitope1. FIG.3A. His-tagged IL-17F epitopes were synthesized to contain strategicscrambling of the sequences either N-terminal or C-terminal to thelocation of the click handle (C48S). 1) SEQ ID NO:39, 2) SEQ ID NO:40,3) SEQ ID NO:41. Scrambled residues are shown in italics. FIG. 3B. PointELISAs for the His-tagged IL-17F epitopes against the two macrocyclicpeptide ligands, PEG₃-biotin-modified Cy(RRATS) (SEQ ID NO:9) andCy(RRAQS) (SEQ ID NO:10), demonstrate preferential binding to thesequence FFQKPES (SEQ ID NO:1).

FIGS. 4A-4C: In vitro characterization of macrocycles developed againstIL-17F Epitope2. FIG. 4A. Sandwich ELISAs for human IL-17F proteinagainst PEG₃-biotin-modified Cy(QKHGP) (SEQ ID NO:15), Cy(TKHGP) (SEQ IDNO:16), Cy(KKGWP) (SEQ ID NO:23), and Cy(RSYNK) (SEQ ID NO:28) yieldEC₅₀ values of 72 to 15 nM. FIG. 4B. Point ELISAs for human IL-17F andIL-17A proteins against the four macrocyclic peptide ligands,PEG₃-biotin-modified Cy(QKHGP) (SEQ ID NO:15), Cy(TKHGP) (SEQ ID NO:16),Cy(KKGWP) (SEQ ID NO:23), and Cy(RSYNK) (SEQ ID NO:28), demonstratepreferential binding to IL-17F. FIG. 4C. Point ELISAs for human IL-17Fand IL-17A proteins against macrocyclic peptide ligands, RRATS (SEQ IDNO:9), RSYNK (SEQ ID NO:28), (QKHGP) (SEQ ID NO:15), demonstratepreferential binding to IL-17F or IL-17A.

FIGS. 5A-5B: 3-D structural representations of the IL-17F homodimer. Thesequences of IL-17F Epitopes1-2 and representative epitope-targetedmacrocycles are overlaid (Tz=triazole). The distances (A) measuredbetween the two epitopes are shown with dashed lines. FIG. 5A. In themonomeric IL-17F protein, the distance between the sequence FFQKPES (SEQID NO:1) (in IL-17F Epitope1) and IL-17F Epitope2, NENQRVS (SEQ ID NO:3)is ˜15 Å. Using a linker whose length is similar to the distance betweenthe two binding sites on the protein, a biligand can be synthesizedcontaining macrocycles targeted to each of the two IL-17F epitopes. FIG.5B. In the homodimeric IL-17F protein, the distance between the sequenceFFQKPES (SEQ ID NO:1) (in IL-17F Epitope1) from one monomer and IL-17FEpitope2, NENQRVS (SEQ ID NO:3) from the other monomer is ˜7 Å. Using alinker whose length is similar to the distance between the two bindingsites bridging the protein dimer, another biligand can be synthesizedcontaining macrocycles targeted to each of the two IL-17F epitopes. PDBID: 1JPY.

FIGS. 6A-6F: Structures of cooperative biligand candidates with linkersranging from 4.4 to 26.4 Å to join the two macrocycles. FIG. 6A.Biotin-PEG₃-Cy(RRATS)(SEQ ID NO:9)-Gly-Cy(QKHGP)(SEQ ID NO:15) (Gly=4.4Å). FIG. 6B. Biotin-PEG₃-Cy(RRATS)(SEQ ID NO:9)-PEG₁-Cy(QKHGP)(SEQ IDNO:15) (PEG₁=8.8 Å). FIG. 6C. Biotin-PEG₃-Cy(RRATS)(SEQ IDNO:9)-PEG₂-Cy(QKHGP)(SEQ ID NO:15) (PEG₂=13.2 Å). FIG. 6D.Biotin-PEG₃-Cy(RRATS)(SEQ ID NO:9)-PEG₃-Cy(QKHGP)(SEQ ID NO:15)(PEG₃=17.6 Å). FIG. 6E. Biotin-PEG₃-Cy(RRATS)(SEQ IDNO:9)-PEG₄-Cy(QKHGP)(SEQ ID NO:15) (PEG₄=22 Å). FIG. 6F.Biotin-PEG₃-Cy(RRATS)(SEQ ID NO:9)-PEG₅-Cy(QKHGP)(SEQ ID NO:15)(PEG₅=26.4 Å).

FIG. 7: Starting point for developing a set of PCC binders against aprotein target. The initial goal is to identify one or more PCCs thatbind to one epitope on the protein target (12), and one or moredifferent PCCs binding to a second epitope (13). Additional PCCs thatbind to a third, fourth, etc., epitope may be useful as well. Theepitope targeted PCC method teaches that this may be accomplished byscreening peptide libraries against synthetic epitopes (SynEps) (14,15). A SynEp is a polypeptide that has the sequence of the naturallyoccurring target epitope, except that one position contains anartificial amino acid that presents an azide or acetylene chemical group(16), called a click handle. The SynEp is further modified to contain anassay handle, such as a biotin group, at the N- or C-terminus (17). Thescreening procedure has been described previously (Das, S. et al., AGeneral Synthetic Approach for Designing Epitope Targeted MacrocyclicPeptide Ligands. Angew. Chem. Int. Ed. Engl. 2015, incorporated hereinby reference in its entirety). Using that procedure, one identifies atleast one unique peptide binder to each of at least two epitopes on thetarget. Those peptide binders are validated via carrying out bindingassays against the full protein target (11) as well as against theSynEps. For those binding assays, the SynEps are prepared with thenaturally occurring residue in place of the click handle (16). Ideally,the different regions of the target protein to which the differentligands bind will be relatively close together (a few nanometers orless) in the tertiary protein structure. For even a single SynEp, ascreen can produce PCCs that bind to two different sites.

FIG. 8: PCC that binds to two different sites. The region representingthe epitope of interest (12) is highlighted against a dimmer backgroundof the full protein (11). The amino acid residue that was substitutedfor a click handle in the SynEp structure is indicated by a star (22).During the SynEp screening steps, PCCs that bind to the N-terminal sideof the epitope (23) or the C-terminal side (24) may both be identified.

FIG. 9: Estimation of optimal linker length. A first PCC (31) that bindsto the N-side of one epitope (12) and a second PCC (32) binding to theC-side of a second epitope (13) are shown. Analysis of this bindingarrangement, together with the structure of the protein from, forexample, the Protein Database, permits an estimate of the length of anoptimized linker (33). Such an estimate can narrow down the choice ofcandidate linkers to a very small number. One example might be to usesuch a length estimate to select one or two length-marched polyethyleneglycol oligomers for testing. The best linker (34) is the one thatbrings the biligand affinity closest to that of a fully cooperativebinder.

FIG. 10: IL-17F and IL-17A are close homologs. Alignment of IL-17F (SEQID NO:38) and IL-17A (SEQ ID NO:37). Residues shaded green areidentical, yellow are homologous and no highlight are unique.

FIG. 11: Generation of ligands for IL-17F and IL-17A. Anchor ligandswere generated against IL-17F using two epitopes and against IL-17Ausing one epitope.

FIGS. 12A-12D: Designing an optimized PCC biligand against IL-17F byexploiting cooperativity. FIG. 12A. Drawing of full-length IL-17F. PCCswere developed against the epitopes labeled L1 and L2. FIG. 12B.Expanded view of IL-17F showing the targeted region. The distancebetween the chemically accessible spots on the PCCs is estimated to be15 Å. FIG. 12C. Demonstration that a linker of length closest to 15 Å(PEG₃) yields the best binder (by ˜10-fold). Biligands are of the formBiotin-PEG₃-Cy(RSYNK)(SEQ ID NO:28)-PEG_(x)-Cy(RRATS)(SEQ ID NO:9) (x=1to 5). FIG. 12D. Graph showing that that a linker of length closest to15 Å (PEG₃) yields the best binder (by ˜10-fold).

FIGS. 13A-13E: Structures of biligands with PEG linkers ranging from 8.8to 26.4 Å to join the two macrocycles. FIG. 13A.Biotin-PEG₃-Cy(RSYNK)(SEQ ID NO:28)-PEG₁-Cy(RRATS)(SEQ ID NO:9)(PEG₁=8.8 Å). FIG. 13B. Biotin-PEG₃-Cy(RSYNK)(SEQ IDNO:28)-PEG₂-Cy(RRATS)(SEQ ID NO:9) (PEG₂=13.2 Å). FIG. 13C.Biotin-PEG₃-Cy(RSYNK)(SEQ ID NO:28)-PEG₃-Cy(RRATS)(SEQ ID NO:9)(PEG₃=17.6 Å). FIG. 13D. Biotin-PEG₃-Cy(RSYNK)(SEQ IDNO:28)-PEG₄-Cy(RRATS)(SEQ ID NO:9) (PEG₄=22 Å). FIG. 13E.Biotin-PEG₃-Cy(RSYNK)(SEQ ID NO:28)-PEG₅-Cy(RRATS)(SEQ ID NO:9)(PEG₅=26.4 Å).

FIG. 14: Plasmodium falciparum Histidine Rich Protein-2 (Pf.HRP-2). Thesequence map of Pf.HRP-2 is shown (SEQ ID NO:42).

FIG. 15: Structure and EC50 data on YKYYR (SEQ ID NO:29) dimer.

FIG. 16: cy(YKYYR)(SEQ ID NO:29)-linker-cy(GWNVDL)(SEQ ID NO:30)biligand with linker developed through library screening.

DETAILED DESCRIPTION

The following description of the invention is merely intended toillustrate various embodiments of the invention. As such, the specificmodifications discussed are not to be construed as limitations on thescope of the invention. It will be apparent to one skilled in the artthat various equivalents, changes, and modifications may be made withoutdeparting from the scope of the invention, and it is understood thatsuch equivalent embodiments are to be included herein.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

Definitions

“Amino” refers to the —NH₂ radical.

“Cyano” refers to the —CN radical.

“Hydroxy” or “hydroxyl” refers to the —OH radical.

“Imino” refers to the ═NH substituent.

“Nitro” refers to the —NO₂ radical.

“Oxo” refers to the ═O substituent.

“Thioxo” refers to the ═S substituent.

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, which is saturated orunsaturated (i.e., contains one or more double and/or triple bonds),having from one to twelve carbon atoms (C₁-C₁₂, alkyl), preferably oneto eight carbon atoms (C₁-C₈ alkyl) or one to six carbon atoms (C₁-C₆alkyl), and which is attached to the rest of the molecule by a singlebond, e.g., methyl, ethyl, n propyl, 1 methylethyl (isopropyl), n-butyl,n-pentyl, 1,1 dimethylethyl (t-butyl), 3 methylhexyl, 2 methylhexyl,ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta 1,4 dienyl,ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unlessstated otherwise specifically in the specification, an alkyl group maybe optionally substituted.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a radical group,consisting solely of carbon and hydrogen, which is saturated orunsaturated (i.e., contains one or more double and/or triple bonds), andhaving from one to twelve carbon atoms, e.g., methylene, ethylene,propylene, n-butylene, ethenylene, propenylene, n-butenylene,propynylene, n-butynylene, and the like. The alkylene chain is attachedto the rest of the molecule through a single or double bond and to theradical group through a single or double bond. The points of attachmentof the alkylene chain to the rest of the molecule and to the radicalgroup can be through one carbon or any two carbons within the chain.Unless stated otherwise specifically in the specification, an alkylenechain may be optionally substituted.

“Alkoxy” refers to a radical of the formula —OR_(a) where R_(a) is analkyl radical as defined above containing one to twelve carbon atoms.Unless stated otherwise specifically in the specification, an alkoxygroup may be optionally substituted.

“Aminocarbonyl” refers to a radical of the formula —C(═O)NR_(a)R_(a),where each R_(a) is independently H, alkyl or a linker moiety.

“α-amino carbonyl” refers to a radical of the formula—C(═O)CR_(b)(NR_(a)R_(a)), where each R_(a) is independently H, alkyl ora linker moiety and R_(b) is H or alkyl. In some embodiments, an alphaamino carbonyl is part of a cyclic moiety (e.g., peptide) where thecarbonyl is within the ring and the amino (NR_(a)R_(a)) is exocyclic.For example, in certain embodiments an alpha aminocarbonyl is useful forEdman degradation of cyclic peptides.

α-amido carbonyl” refers to a radical of the formula—C(═O)CR_(b)(N(C═O)R_(a)R_(a)), where each R_(a) is independently H,alkyl or a linker moiety and R_(b) is H or alkyl. In some embodiments,an alpha amido carbonyl is part of a cyclic moiety (e.g., peptide) wherethe carbonyl is within the ring and the amido (N(C═O)R_(a)R_(a)) isexocyclic.

“Alkylamino” refers to a radical of the formula —NHR_(a) or —NR_(a)R_(a)where each R_(a) is, independently, an alkyl radical as defined abovecontaining one to twelve carbon atoms. Unless stated otherwisespecifically in the specification, an alkylamino group may be optionallysubstituted.

“Thioalkyl” refers to a radical of the formula —SR_(a) where R_(a) is analkyl radical as defined above containing one to twelve carbon atoms.Unless stated otherwise specifically in the specification, a thioalkylgroup may be optionally substituted.

“Aryl” refers to a hydrocarbon ring system radical comprising hydrogen,6 to 18 carbon atoms and at least one aromatic ring. For purposes ofthis invention, the aryl radical may be a monocyclic, bicyclic,tricyclic or tetracyclic ring system, which may include fused or bridgedring systems. Aryl radicals include, but are not limited to, arylradicals derived from aceanthrylene, acenaphthylene, acephenanthrylene,anthracene, azulene, benzene, chrysene, fluoranthene, fluorene,as-indacene, s indacene, indane, indene, naphthalene, phenalene,phenanthrene, pleiadene, pyrene, and triphenylene. Unless statedotherwise specifically in the specification, the term “aryl” or theprefix “ar-” (such as in “aralkyl”) is meant to include aryl radicalsthat are optionally substituted.

“Aralkyl” refers to a radical of the formula —R_(b)—R_(e) where R_(b) isan alkylene chain as defined above and R_(c) is one or more arylradicals as defined above, for example, benzyl, diphenylmethyl and thelike. Unless stated otherwise specifically in the specification, anaralkyl group may be optionally substituted.

“Cycloalkyl” or “carbocyclic ring” refers to a stable non aromaticmonocyclic or polycyclic hydrocarbon radical consisting solely of carbonand hydrogen atoms, which may include fused or bridged ring systems,having from three to fifteen carbon atoms, preferably having from threeto ten carbon atoms, and which is saturated or unsaturated and attachedto the rest of the molecule by a single bond. Monocyclic radicalsinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example,adamantyl, norbornyl, decalinyl, 7,7 dimethyl bicyclo[2.2.1]heptanyl,and the like. Unless otherwise stated specifically in the specification,a cycloalkyl group may be optionally substituted.

“Cycloalkylalkyl” refers to a radical of the formula —R_(b)R_(d) whereR_(b) is an alkylene chain as defined above and R_(d) is a cycloalkylradical as defined above. Unless stated otherwise specifically in thespecification, a cycloalkylalkyl group may be optionally substituted.

“Fused” refers to any ring structure described herein which is fused toan existing ring structure in the compounds of the invention. When thefused ring is a heterocyclyl ring or a heteroaryl ring, any carbon atomon the existing ring structure which becomes part of the fusedheterocyclyl ring or the fused heteroaryl ring may be replaced with anitrogen atom.

“Halo” or “halogen” refers to bromo, chloro, fluoro or iodo.

“Haloalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more halo radicals, as defined above, e.g.,trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2 trifluoroethyl,1,2 difluoroethyl, 3 bromo 2 fluoropropyl, 1,2 dibromoethyl, and thelike. Unless stated otherwise specifically in the specification, ahaloalkyl group may be optionally substituted.

“Heterocyclyl” or “heterocyclic ring” refers to a stable 3 to 18membered non aromatic ring radical which consists of two to twelvecarbon atoms and from one to six heteroatoms selected from the groupconsisting of nitrogen, oxygen and sulfur. Unless stated otherwisespecifically in the specification, the heterocyclyl radical may be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, which mayinclude fused or bridged ring systems; and the nitrogen, carbon orsulfur atoms in the heterocyclyl radical may be optionally oxidized; thenitrogen atom may be optionally quaternized; and the heterocyclylradical may be partially or fully saturated. Examples of suchheterocyclyl radicals include, but are not limited to, dioxolanyl,thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl,imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl,octahydroindolyl, octahydroisoindolyl, 2 oxopiperazinyl, 2oxopiperidinyl, 2 oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4 piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,thiomorpholinyl, thiamorpholinyl, 1 oxo thiomorpholinyl, and 1,1 dioxothiomorpholinyl. Unless stated otherwise specifically in thespecification, a heterocyclyl group may be optionally substituted.

“N-heterocyclyl” refers to a heterocyclyl radical as defined abovecontaining at least one nitrogen and where the point of attachment ofthe heterocyclyl radical to the rest of the molecule is through anitrogen atom in the heterocyclyl radical. Unless stated otherwisespecifically in the specification, a N-heterocyclyl group may beoptionally substituted.

“Heterocyclylalkyl” refers to a radical of the formula —RbRe where R_(b)is an alkylene chain as defined above and Re is a heterocyclyl radicalas defined above, and if the heterocyclyl is a nitrogen containingheterocyclyl, the heterocyclyl may be attached to the alkyl radical atthe nitrogen atom. Unless stated otherwise specifically in thespecification, a heterocyclylalkyl group may be optionally substituted.

“Heteroaryl” refers to a 5 to 14 membered ring system radical comprisinghydrogen atoms, one to thirteen carbon atoms, one to six heteroatomsselected from the group consisting of nitrogen, oxygen and sulfur, andat least one aromatic ring. For purposes of this invention, theheteroaryl radical may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heteroarylradical may be optionally oxidized; the nitrogen atom may be optionallyquaternized. Examples include, but are not limited to, azepinyl,acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl,benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxepinyl, 1,4 benzodioxanyl, benzonaphthofuranyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2 a]pyridinyl,carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl,furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl,isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl,isoxazolyl, naphthyridinyl, oxadiazolyl, 2 oxoazepinyl, oxazolyl,oxiranyl, 1-oxidopyridinyl, 1 oxidopyrimidinyl, 1-oxidopyrazinyl,1-oxidopyridazinyl, 1 phenyl 1H pyrrolyl, phenazinyl, phenothiazinyl,phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl,pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl,quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl,tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl,triazinyl, and thiophenyl (i.e. thienyl). Unless stated otherwisespecifically in the specification, a heteroaryl group may be optionallysubstituted.

“N-heteroaryl” refers to a heteroaryl radical as defined abovecontaining at least one nitrogen and where the point of attachment ofthe heteroaryl radical to the rest of the molecule is through a nitrogenatom in the heteroaryl radical. Unless stated otherwise specifically inthe specification, an N-heteroaryl group may be optionally substituted.

“Heteroarylalkyl” refers to a radical of the formula —R_(b)R_(f) whereR_(b) is an alkylene chain as defined above and Rf is a heteroarylradical as defined above. Unless stated otherwise specifically in thespecification, a heteroarylalkyl group may be optionally substituted.

The term “substituted” used herein means any of the above groups (e.g.,alkyl, alkylene, alkoxy, alkylamino, aminocarbonyl, α-aminocarbonyl,α-amidocarbonyl, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl,haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl,N-heteroaryl and/or heteroarylalkyl) wherein at least one hydrogen atomis replaced by a bond to a non-hydrogen atoms such as, but not limitedto: a halogen atom such as F, Cl, Br, and I; an oxygen atom in groupssuch as hydroxyl groups, alkoxy groups, and ester groups; a sulfur atomin groups such as thiol groups, thioalkyl groups, sulfone groups,sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such asamines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines,diarylamines, N-oxides, imides, and enamines; a silicon atom in groupssuch as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilylgroups, and triarylsilyl groups; and other heteroatoms in various othergroups. “Substituted” also means any of the above groups in which one ormore hydrogen atoms are replaced by a higher-order bond (e.g., a double-or triple-bond) to a heteroatom such as oxygen in oxo, carbonyl,carboxyl, and ester groups; and nitrogen in groups such as imines,oximes, hydrazones, and nitriles. For example, “substituted” includesany of the above groups in which one or more hydrogen atoms are replacedwith —NR_(g)R_(h), —NR_(g)C(═O)R_(h), —NR_(g)C(═O)NR_(g)R_(h),—NR_(g)C(═O)OR_(h), —NR_(g)SO2R_(h), —OC(═O) NR_(g)R_(h), —OR_(g),—SR_(g), —SOR_(g), —SO₂R_(g), —OSO₂R_(g), —SO₂OR_(g), ═NSO₂R_(g), and—SO₂NR_(g)R_(h). “Substituted” also means any of the above groups inwhich one or more hydrogen atoms are replaced with —C(═O)R_(g),—C(═O)OR_(g), —C(═O)NR_(g)R_(h), —CH₂SO₂R_(g), —CH₂SO₂NR_(g)R_(h). Inthe foregoing, R_(g) and R_(h) are the same or different andindependently hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl,aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl,N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/orheteroarylalkyl. “Substituted” further means any of the above groups inwhich one or more hydrogen atoms are replaced by a bond to an amino,cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkoxy,alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl,haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl,N-heteroaryl and/or heteroarylalkyl group. In addition, each of theforegoing substituents may also be optionally substituted with one ormore of the above substituents.

“Prodrug” is meant to indicate a compound that may be converted underphysiological conditions or by solvolysis to a biologically activecompound of the invention (i.e., a disclosed capture agent). Thus, theterm “prodrug” refers to a metabolic precursor of a compound of theinvention that is pharmaceutically acceptable. A prodrug may be inactivewhen administered to a subject in need thereof, but is converted in vivoto an active compound of the invention. Prodrugs are typically rapidlytransformed in vivo to yield the parent compound of the invention, forexample, by hydrolysis in blood. The prodrug compound often offersadvantages of solubility, tissue compatibility or delayed release in amammalian organism (see, Bundgard, H., Design of Prodrugs (1985), pp. 79, 21 24 (Elsevier, Amsterdam)). A discussion of prodrugs is provided inHiguchi, T., et al., A.C.S. Symposium Series, Vol. 14, and inBioreversible Carriers in Drug Design, Ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987.

The term “prodrug” is also meant to include any covalently bondedcarriers, which release the active compound of the invention in vivowhen such prodrug is administered to a mammalian subject. Prodrugs of acompound of the invention may be prepared by modifying functional groupspresent in the compound of the invention in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe parent compound of the invention. Prodrugs include compounds of theinvention wherein a hydroxy, amino or mercapto group is bonded to anygroup that, when the prodrug of the compound of the invention isadministered to a mammalian subject, cleaves to form a free hydroxy,free amino or free mercapto group, respectively. Examples of prodrugsinclude, but are not limited to, acetate, formate and benzoatederivatives of alcohol or amide derivatives of amine functional groupsin the compounds of the invention and the like.

The invention disclosed herein is also meant to encompass allpharmaceutically acceptable disclosed capture agents beingisotopically-labelled by having one or more atoms replaced by an atomhaving a different atomic mass or mass number. Examples of isotopes thatcan be incorporated into the disclosed compounds include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, andiodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³¹P,³²P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I, and ¹²⁵I, respectively. These radiolabelledcompounds could be useful to help determine or measure the effectivenessof the compounds, by characterizing, for example, the site or mode ofaction, or binding affinity to pharmacologically important site ofaction. Certain isotopically-labelled disclosed capture agents, forexample, those incorporating a radioactive isotope, are useful in drugand/or substrate tissue distribution studies. The radioactive isotopestritium, i.e. ³H, and carbon-14, i.e. ¹⁴C, are particularly useful forthis purpose in view of their ease of incorporation and ready means ofdetection.

Substitution with heavier isotopes such as deuterium, i.e. ²H, mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining substrate receptor occupancy. Isotopically-labeled captureagents can generally be prepared by conventional techniques known tothose skilled in the art or by processes analogous to those described inthe Preparations and Examples as set out below using an appropriateisotopically-labeled reagent in place of the non-labeled reagentpreviously employed.

The invention disclosed herein is also meant to encompass the in vivometabolic products of the disclosed capture agents. Such products mayresult from, for example, the oxidation, reduction, hydrolysis,amidation, esterification, and the like of the administered compound,primarily due to enzymatic processes. Accordingly, the inventionincludes compounds produced by a process comprising administering acompound of this invention to a mammal for a period of time sufficientto yield a metabolic product thereof. Such products are typicallyidentified by administering a radiolabelled compound of the invention ina detectable dose to an animal, such as rat, mouse, guinea pig, monkey,or to human, allowing sufficient time for metabolism to occur, andisolating its conversion products from the urine, blood or otherbiological samples.

“Mammal” includes humans and both domestic animals such as laboratoryanimals and household pets (e.g., cats, dogs, swine, cattle, sheep,goats, horses, rabbits), and non-domestic animals such as wildlife andthe like.

“Optional” or “optionally” means that the subsequently described eventof circumstances may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. For example, “optionally substituted aryl” means that thearyl radical may or may not be substituted and that the descriptionincludes both substituted aryl radicals and aryl radicals having nosubstitution.

“Pharmaceutically acceptable carrier, diluent or excipient” includeswithout limitation any adjuvant, carrier, excipient, glidant, sweeteningagent, diluent, preservative, dye/colorant, flavor enhancer, surfactant,wetting agent, dispersing agent, suspending agent, stabilizer, isotonicagent, solvent, or emulsifier which has been approved by the UnitedStates Food and Drug Administration as being acceptable for use inhumans or domestic animals.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but are not limited to,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroaceticacid, undecylenic acid, and the like.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Preferred inorganic salts are the ammonium, sodium, potassium, calcium,and magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as ammonia,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol, 2dimethylaminoethanol, 2 diethylaminoethanol, dicyclohexylamine, lysine,arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine,benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine,theobromine, triethanolamine, tromethamine, purines, piperazine,piperidine, N ethylpiperidine, polyamine resins and the like.Particularly preferred organic bases are isopropylamine, diethylamine,ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

The compounds (capture agents) of the invention, or theirpharmaceutically acceptable salts may contain one or more asymmetriccenters and may thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R) or (S) or, as (D) or (L) for amino acids. Thepresent invention is meant to include all such possible isomers, as wellas their racemic and optically pure forms. Optically active (+) and ( )(R) and (S), or (D) and (L) isomers may be prepared using chiralsynthons or chiral reagents, or resolved using conventional techniques,for example, chromatography and fractional crystallization. Conventionaltechniques for the preparation/isolation of individual enantiomersinclude chiral synthesis from a suitable optically pure precursor orresolution of the racemate (or the racemate of a salt or derivative)using, for example, chiral high pressure liquid chromatography (HPLC).When the compounds described herein contain olefinic double bonds orother centers of geometric asymmetry, and unless specified otherwise, itis intended that the compounds include both E and Z geometric isomers.Likewise, all tautomeric forms are also intended to be included.(D)-amino acids (also referred to as D-amino acids) are referred toherein in lower case letters (e.g. D-valine is referred to as “v”),while (L)-amino acids (also referred to herein as L-amino acids) arereferred to in upper case letters (e.g. L-valine or valine is referredto as “V”). Glycine is non-chiral and is referred to as “G”.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. The present invention contemplatesvarious stereoisomers and mixtures thereof and includes “enantiomers”,which refers to two stereoisomers whose molecules are nonsuperimposeablemirror images of one another.

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. The present invention includestautomers of any said compounds.

Often crystallizations produce a solvate of the compound of theinvention. As used herein, the term “solvate” refers to an aggregatethat comprises one or more molecules of a compound of the invention withone or more molecules of solvent. The solvent may be water, in whichcase the solvate may be a hydrate. Alternatively, the solvent may be anorganic solvent. Thus, the compounds of the present invention may existas a hydrate, including a monohydrate, dihydrate, hemihydrate,sesquihydrate, trihydrate, tetrahydrate and the like, as well as thecorresponding solvated forms. The compound of the invention may be truesolvates, while in other cases, the compound of the invention may merelyretain adventitious water or be a mixture of water plus someadventitious solvent.

The term “capture agent” as used herein refers to a composition thatcomprises two or more target-binding moieties and which specificallybinds to a target protein via those target-binding moieties. Eachtarget-binding moiety exhibits binding affinity for the target protein,either individually or in combination with other target-bindingmoieties. In certain embodiments, each target-binding moiety binds tothe target protein via one or more non-covalent interactions, includingfor example hydrogen bonds, hydrophobic interactions, and van der Waalsinteractions. A capture agent may comprise one or more organicmolecules, including for example polypeptides, peptides,polynucleotides, and other non-polymeric molecules. In some aspects acapture agent is a protein catalyzed capture agent (PCC).

The term “epitope” as used herein refers to a distinct molecular surfaceof a protein (e.g., IL-17F). Typically, the epitope is a polypeptide andit can act on its own as a finite sequence of 10-40 amino acids.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to an amino acid sequence comprising apolymer of amino acid residues. The terms apply to amino acid polymersin which one or more amino acid residues is an artificial chemicalmimetic of a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers and non-naturally occurringamino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids, andisomers thereof. Naturally occurring amino acids are those encoded bythe genetic code, as well as those amino acids that are later modified,e.g., hydroxyproline, carboxyglutamate, O-phosphoserine, and isomersthereof. The term “amino acid analogs” refers to compounds that have thesame basic chemical structure as a naturally occurring amino acid, i.e.,a carbon that is bound to a hydrogen, a carboxyl group, an amino group,and an R group, e.g., homoserine, norleucine, methionine sulfoxide,methionine methyl sulfonium. Such analogs have modified R groups (e.g.,norleucine) or modified peptide backbones, but retain the same basicchemical structure as a naturally occurring amino acid. The term “aminoacid mimetics” refers to chemical compounds that have a structure thatis different from the general chemical structure of an amino acid, butthat functions in a manner similar to a naturally occurring amino acid.Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission.

The term “non-natural amino acid” as used herein refers to an amino acidthat is different from the twenty naturally occurring amino acids(alanine, arginine, glycine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, serine, threonine, histidine, lysine,methionine, proline, valine, isoleucine, leucine, tyrosine, tryptophan,phenylalanine) in its side chain functionality. The non-natural aminoacid can be a close analog of one of the twenty natural amino acids, orit can introduce a completely new functionality and chemistry, as longas the hydrophobicity of the non-natural amino acid is either equivalentto or greater than that of the natural amino acid. The non-natural aminoacid can either replace an existing amino acid in a protein(substitution), or be an addition to the wild type sequence (insertion).The incorporation of non-natural amino acids can be accomplished byknown chemical methods including solid-phase peptide synthesis or nativechemical ligation, or by biological methods.

The terms “specific binding,” “selective binding,” “selectively binds,”or “specifically binds” as used herein refer to capture agent binding toan epitope on a predetermined antigen. Typically, the capture agentbinds with an affinity (K_(D)) of approximately less than 10⁻⁷ M, suchas approximately less than 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower.

The term “K_(D)” as used herein refers to the dissociation equilibriumconstant of a particular capture agent-antigen interaction. Typically,the capture agents of the invention bind to IL-17 with a dissociationequilibrium constant (K_(D)) of less than approximately 10⁻⁶ M, 10⁻⁷ M,such as less than approximately 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower,for example, as determined using surface plasmon resonance (SPR)technology in a Biacore instrument using the antigen as the ligand andthe capture agent as the analyte, and binds to the predetermined antigenwith an affinity corresponding to a K_(D) that is at least ten-foldlower, such as at least 100 fold lower, for instance at least 1000 foldlower, such as at least 10,000 fold lower, for instance at least 100,000fold lower than its affinity for binding to a non-specific antigen(e.g., BSA, casein) other than the predetermined antigen or aclosely-related antigen. The amount with which the affinity is lower isdependent on the K_(D) of the capture agent, so that when the K_(D) ofthe capture agent is very low (that is, the capture agent is highlyspecific), then the amount with which the affinity for the antigen islower than the affinity for a non-specific antigen may be at least10,000 fold.

The term “k_(d)” (sec⁻¹) as used herein refers to the dissociation rateconstant of a particular capture agent-antigen interaction. Said valueis also referred to as the K_(off) value.

The term “k_(a)” (M⁻¹×sec⁻¹) as used herein refers to the associationrate constant of a particular capture agent-antigen interaction.

The term “K_(D)” (M) as used herein refers to the dissociationequilibrium constant of a particular capture agent-antigen interaction.

The term “K_(A)” (M⁻¹) as used herein refers to the associationequilibrium constant of a particular capture agent-antigen interactionand is obtained by dividing the k_(a) by the k_(d).

A “pharmaceutical composition” refers to a formulation of a compound ofthe invention and a medium generally accepted in the art for thedelivery of the biologically active compound to mammals, e.g., humans.Such a medium includes all pharmaceutically acceptable carriers,diluents or excipients therefor.

The term “condition” as used herein refers generally to a disease,event, or a change in health status. A change in health status may beassociated with a particular disease or event, in which case the changemay occur simultaneously with or in advance of the disease or event. Inthose cases where the change in health status occurs in advance of adisease or event, the change in health status may serve as a predictorof the disease or event. For example, a change in health status may bean alteration in the expression level of a particular gene associatedwith a disease or event. Alternatively, a change in health status maynot be associated with a particular disease or event.

The terms “treat,” “treating,” or “treatment” as used herein generallyrefer to preventing a condition or event, slowing the onset or rate ofdevelopment of a condition or delaying the occurrence of an event,reducing the risk of developing a condition or experiencing an event,preventing or delaying the development of symptoms associated with acondition or event, reducing or ending symptoms associated with acondition or event, generating a complete or partial regression of acondition, lessening the severity of a condition or event, or somecombination thereof.

An “effective amount” or “therapeutically effective amount” as usedherein refers to an amount effective, at dosages and for periods of timenecessary, to achieve a desired therapeutic result. A therapeuticallyeffective amount of a disclosed capture agent may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the capture agent to elicit a desiredresponse in the individual.

The term “stable” as used herein with regard to a capture agent proteincatalyzed capture agent or pharmaceutical formulation thereof refers tothe agent or formulation retaining structural and functional integrityfor a sufficient period of time to be utilized in the methods describedherein.

The term “synthetic” as used herein with regard to a protein catalyzedcapture agent or capture agent refers to the capture agent has beengenerated by chemical rather than biological means.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using the BLAST 2.0 suite of programsusing default parameters (Altschul, et al., (1997) Nucleic Acids Res.25:3389-402).

As those of ordinary skill in the art will understand, BLAST searchesassume that proteins can be modeled as random sequences. However, manyreal proteins comprise regions of nonrandom sequences, which may behomopolymeric tracts, short-period repeats, or regions enriched in oneor more amino acids. Such low-complexity regions may be aligned betweenunrelated proteins even though other regions of the protein are entirelydissimilar. A number of low-complexity filter programs can be employedto reduce such low-complexity alignments. For example, the SEG (Wootenand Federhen, (1993) Comput. Chem. 17:149-63) and XNU (Claverie andStates, (1993) Comput. Chem. 17:191-201) low-complexity filters can beemployed alone or in combination.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences includes reference to the residuesin the two sequences, which are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. Where sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences, which differ by suchconservative substitutions, are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., according to the algorithm of Meyersand Miller, (1988) Computer Applic. Biol. Sci. 4:11-17, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

The term “substantial identity” or “substantially identical” ofpolynucleotide sequences means that a polynucleotide comprises asequence that has between 50-100% sequence identity, preferably at least50% sequence identity, preferably at least 60% sequence identity,preferably at least 70%, more preferably at least 80%, more preferablyat least 90% and most preferably at least 95%, compared to a referencesequence using one of the alignment programs described using standardparameters. One of skill will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning and thelike. Substantial identity of amino acid sequences for these purposesnormally means sequence identity of between 55-100%, preferably at least55%, preferably at least 60%, more preferably at least 70%, 80%, 90% andmost preferably at least 95%.

In certain embodiments, the term “IL-17F” as used herein refers to humanIL-17F. In some embodiments, IL-17 comprises the following amino acidsequence or an amino acid sequence substantially identical to it.

(SEQ ID NO: 31) 1 MTVKTLHGPA MVKYLLLSIL GLAFLSEAAA RKIPKVGHTFFQKPESCPPV PGGSMKLDIG 61 IINENQRVSM SRNIESRSTS PWNYTVTWDP NRYPSEVVQAQCRNLGCINA QGKEDISMNS 121 VPIQQETLVV RRKHQGCSVS FQLEKVLVTV GCTCVTPVIHRVQ

In other embodiments, IL-17F is a protein encoded by the generepresented by Entrez Gene ID Number 112744.

Development of IL-17F Capture Agents

Antibodies are currently the default detection agent for use indiagnostic platforms. However, antibodies possess several disadvantages,including high cost, poor stability, and, in many cases, lack of propercharacterization and high specificity. The ideal replacement for use indiagnostic assays should be synthetic, stable to a range of thermal andchemical conditions, and display high affinity and specificity for thetarget of interest.

A high quality monoclonal antibody possesses low-nanomolar affinity andhigh target specificity. Interestingly, structural and genetic analysesof the antigen recognition surface have shown that the majority of themolecular diversity of the variable loops is contained in a singlehighly variable loop (CDR-H3). In humans, this loop ranges in size from1-35 residues (15 on average), can adopt a wide range of structuralconformations, and is responsible for most of the interactions with theantigen. The other five loops are significantly less diverse and adoptonly a handful of conformations. This suggests that a carefully selected“anchor” peptide can dominate the mode and strength of the interactionbetween a capture agent and its target protein. It also suggests thatother peptide components, while providing only modest contributions tothe total interaction energy, can supply important scaffolding featuresand specificity elements.

In situ click chemistry is a technique in which a small moleculeenzymatic inhibitor is separated into two moieties, each of which isthen expanded into a small library—one containing acetylenefunctionalities, and the other containing azide groups. The enzymeitself then assembles the ‘best fit’ inhibitor from these librarycomponents by selectively promoting 1,3-dipolar cycloaddition betweenthe acetylene and azide groups to form a triazole linkage (the ‘click’reaction). The protein effectively plays the role of an extremelyselective variant of the Cu(I) catalyst that is commonly used for suchcouplings. The enzyme promotes the click reaction only between thoselibrary components that bind to the protein in the right orientation.The resultant inhibitor can exhibit far superior affinitycharacteristics relative to the initial inhibitor that formed the basisof the two libraries.

Sequential in situ click chemistry extends the in situ click chemistryconcept to enable the discovery of multiligand capture agents (see: USSN20100009896, incorporated herein by reference). This process was usedpreviously to produce a triligand capture agent against the modelprotein carbonic anhydrase II (CAII). Sequential in situ click chemistryhas several advantages. First, structural information about the proteintarget is replaced by the ability to sample a very large chemical spaceto identify the ligand components of the capture agent. For example, aninitial ligand may be identified by screening the protein against alarge (>106 element) one-bead-one-compound (OBOC) peptide library, wherethe peptides themselves may be comprised of natural, non-natural, and/orartificial amino acids. The resultant anchor ligand is then utilized inan in situ click screen, again using a large OBOC library, to identify abiligand binder. A second advantage is that the process can be repeated,so that the biligand is used as an anchor to identify a triligand, andso forth. The final capture agent can then be scaled up using relativelysimple and largely automated chemistries, and it can be developed with alabel, such as a biotin group, as an intrinsic part of its structure.This approach permits the exploration of branched, cyclic, and linearcapture agent architectures. While many strategies for protein-directedmultiligand assembly have been described, most require detailedstructural information on the target to guide the screening strategy,and most (such as the original in situ click approach), are optimizedfor low-diversity small molecule libraries.

The present embodiment further generalizes the in situ click applicationto naively find an anchor ligand using in situ click. In previousapproaches, a known binder was necessary to begin the ligand. Thismethod provides a mechanism to find an anchor ligand de novo.

As described herein, an iterative in situ click chemistry approach wasutilized to synthesize a biligand capture agent that specifically bindsIL-17F. This in situ click chemistry approach comprised two steps.First, two “anchor” ligands were found that bound IL-17F at distinct butrelatively close sites. Second, a linker of an appropriate size wasfound that bound the two ligands producing a capture agent with higheraffinity for IL-17F.

The capture agents generated by the methods disclosed herein were foundto display binding affinity for IL-17F. The capture agents were shown tofunction as both capture and detection agents in ELISA assays andefficiently immunoprecipitate IL-17F.

IL-17F Capture Agents

In one aspect, provided herein is a stable, synthetic capture agent thatspecifically binds IL-17F, wherein the capture agent comprises two ormore “anchor” ligands (also referred to as simply “ligands” herein) anda linker and wherein the ligands selectively bind IL-17F.

In certain embodiments, a ligand comprises one or more polypeptides orpeptides. In certain of these embodiments, a target-binding moietycomprises one or more peptides comprising D-amino acids, L-amino acids,and/or amino acids substituted with functional groups selected from thegroup consisting of substituted and unsubstituted alkyl, substituted andunsubstituted azido, substituted and unsubstituted alkynyl, substitutedand unsubstituted biotinyl, substituted and unsubstituted azioalkyl,substituted and unsubstituted polyethyleneglycolyl, and substituted andunsubstituted 1,2,3-triazole.

In certain embodiments, the ligands are linked to one another via acovalent linkage through a linker. In certain of these embodiments, theligand and linker are linked to one another via an amide bond or a1,4-disubstituted-1,2,3-triazole linkage as shown below:

In those embodiments where the ligands and linker are linked to oneanother via a 1,4-disubstituted-1,2,3-triazole linkage, the1,4-disubstituted-1,2,3-triazole linkage may be formed by Cu-CatalyzedAzide/Alkyne Cycloaddition (CuAAC).

In certain embodiments, the ligands and linker are linked to one anotherby a Tz4 linkage having the following structure:

In certain embodiments, the ligands and linker are linked to one anotherby a Tz5 linkage having the following structure:

In those embodiments wherein one or more of the ligands and linker arelinked to one another via amide bonds, the amide bond may be formed bycoupling a carboxylic acid group and an amine group in the presence of acoupling agent (e.g., 0-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU),N-hydroxy-7-aza-benzotriazole (HOAt), or diisopropylethylamine (DIEA) inDMF).

In certain embodiments, the capture agents provided herein are stableacross a range of reaction conditions and/or storage times. A captureagent that is “stable” as used herein maintains the ability tospecifically bind to a target protein. In certain embodiments, thecapture agents provided herein are more stable than an antibody bindingto the same target protein under one or more reaction and/or storageconditions. For example, in certain embodiments the capture agentsprovided herein are more resistant to proteolytic degradation than anantibody binding to the same target protein.

In certain embodiments, the capture agents provided herein have ashelf-life of greater than six months, meaning that they are stable instorage for greater than six months. In certain of these embodiments,the capture agents have a shelf-life of one year or greater, two yearsor greater, or more than three years. In certain of these embodiments,the capture agents are stored as a lyophilized powder. In certainembodiments, the capture agents provided herein have a longer shelf-lifethan an antibody binding to the same target protein.

In certain embodiments, the capture agents provided herein are stable attemperatures ranging from about −80° to about 120° C. In certain ofthese embodiments, the capture agents are stable within a temperaturerange of −80° to −40° C.; −40° to −20° C.; −20° to 0° C.; 0° to 20° C.;20° to 40° C.; 40° to 60° C.; 60° to 80° C.; and/or 80° to 120° C. Incertain embodiments, the capture agents provided herein are stableacross a wider range of temperatures than an antibody binding to thesame target protein, and/or remain stable at a specific temperature fora longer time period than an antibody binding to the same targetprotein.

In certain embodiments, the capture agents provided herein are stable ata pH range from about 3.0 to about 8.0. In certain embodiments, therange is about 4.0 to about 7.0. In certain embodiments, the range isabout 7.0 to about 8.0.

In certain embodiments, the capture agents provided herein are stable inhuman serum for more than 12 hours. In certain of these embodiments, thecapture agents are stable in human serum for more than 18 hours, morethan 24 hours, more than 36 hours, or more than 48 hours. In certainembodiments, the capture agents provided herein are stable for a longerperiod of time in human serum than an antibody binding to the sametarget protein. In certain embodiments, the capture agents are stable asa powder for two months at a temperature of about 60° C.

In certain embodiments, the capture agents provided herein may compriseone or more detection labels, including for example biotin,copper-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(copper-DOTA), ⁶⁴Cu DOTA, ⁶⁸Ga DOTA, ¹⁸F, ⁶⁴Cu, ⁶⁸Ga, ⁸⁹Zr, ¹²⁴I, ⁸⁶Y,^(94m)Tc, ^(110m)In, 11C, ⁷⁶Br, ¹²³I, ¹³¹I, ⁶⁷Ga, ¹¹¹In and ^(99m)Tc, orother radiolabeled products that may include gamma emitters, protonemitters, positron emitters, tritium, or covered tags detectable byother methods (i.e., gadolinium) among others. In a particularembodiment, the detection label is ¹⁸F. In certain embodiments, thecapture agents may be modified to be used as imaging agents. The imagingagents may be used as diagnostic agents.

In certain embodiments, the capture agents provided herein may bemodified to obtain a desired chemical or biological activity. Examplesof desired chemical or biological activities include, withoutlimitation, improved solubility, stability, bioavailability,detectability, or reactivity. Examples of specific modifications thatmay be introduced to a capture agent include, but are not limited to,cyclizing the capture agent through formation of a disulfide bond;modifying the capture agent with other functional groups or molecules.Similarly, a capture agent may be synthesized to bind to non-canonicalor non-biological epitopes on proteins, thereby increasing theirversatility. In certain embodiments, the capture agent may be modifiedby modifying the synthesis blocks of the target-binding moieties beforethe coupling reaction.

Methods of Making/Screening Capture Agents

Provided herein in certain embodiments are methods of screeningtarget-binding moieties and/or making capture agents that comprise thesetarget-binding moieties. Methods for screening target-binding moietiesand/or making capture agents that comprise these target-binding moietiescan also be found in International Publication Nos. WO 2012/106671, WO2013/033561, WO 2013/009869 and WO 2014/074907, each of which isincorporated by reference, herein, in their entireties.

In certain embodiments, two separately-identified ligands that bind totwo different regions of the same protein (the target) are chemicallylinked together to form a biligand. By optimizing a linker of the twoligands, the biligand formed by the ligands and linker can exhibit abinding affinity that is far superior to either of the individualligands. This enhanced binding effect is called binding cooperativity.For an ideal cooperative binder, the thermodynamic binding energies ofthe individual ligands to the target will sum to yield the bindingenergy of the linked biligand. This means that the binding affinityconstant (K_(D)) of the linked biligand will be the product of thebinding affinity of the individual ligands (i.e. K_(D)=K_(D1)×K_(D2),where the subscripts 1 and 2 refer to the two ligands). In practice,full cooperative binding is rarely, if ever, achieved. Thus, acomparison of the properties of a linked biligand against those of afully cooperative binder provides a measurement of how optimally the twoligands were linked.

If the protein target has a known and well-defined tertiary (folded)structure, then key aspects of this targeting method involve strategiesfor identifying ligands that bind to preferred regions of the protein,followed by approaches for identifying an optimized linker. If theprotein does not have a well-defined tertiary structure, the disclosuredescribes strategies designed to still achieve a significant measure ofcooperative binding from a biligand.

FIG. 7 describes the starting point for developing a set of PCC bindersagainst a protein target (11). The initial goal is to identify one ormore PCCs that bind to one epitope on the protein target (12), and oneor more different PCCs binding to a second epitope (13). Additional PCCsthat bind to a third, fourth, etc., epitope may be useful as well. Theepitope targeted PCC method teaches that this may be accomplished byscreening peptide libraries against synthetic epitopes (SynEps) such asthose shown in FIG. 7 (14, 15). A SynEp is a polypeptide that has thesequence of the naturally occurring target epitope, except that oneposition contains an artificial amino acid that presents an azide oracetylene chemical group (16), called a click handle. The SynEp isfurther modified to contain an assay handle, such as a biotin group, atthe N- or C-terminus (17). The screening procedure can be done using anyprocedure disclosed herein or known in the art. By screening, oneidentifies at least one unique peptide binder to each of at least twoepitopes on the target. Those peptide binders are validated via carryingout binding assays against the full protein target (11) as well asagainst the SynEps. For those binding assays, the SynEps are preparedwith the naturally occurring residue in place of the click handle (16).

Ideally, the different regions of the target protein to which thedifferent ligands bind will be relatively close together (a fewnanometers or less) in the tertiary protein structure. For even a singleSynEp, a screen can produce PCCs that bind to two different sites. InFIG. 8, the region representing the epitope of interest (12) ishighlighted against a dimmer background of the full protein (11). Theamino acid residue that was substituted for a click handle in the SynEpstructure is indicated by a star (22). During the SynEp screening steps,PCCs that bind to the N-terminal side of the epitope (23) or theC-terminal side (24) may both be identified.

Once the epitope targeted PCCs are identified, there are several methodsfor selecting a linker.

In a first embodiment, if the folded structure of the protein is known,and if the PCCs bind to that folded structure, then one can use thatinformation, plus knowledge of which PCCs bind to which epitopes, toestimate an optimal linker length. This is illustrated in FIG. 9. Thisfigure shows one PCC (31) that binds to the N-side of one epitope (12)and a second PCC (32) binding to the C-side of a second epitope (13).Analysis of this binding arrangement, together with the structure of theprotein from, for example, the Protein Database, permits an estimate ofthe length of an optimized linker (33). Such an estimate can narrow downthe choice of candidate linkers to a very small number. One examplemight be to use such a length estimate to select one or twolength-matched polyethylene glycol oligomers for testing. The bestlinker (34) is the one that brings the biligand affinity closest to thata fully cooperative binder.

In a second embodiment, if the folded structure of the protein is notknown, or if the protein simply does not have a well-defined foldedstructure, then one uses as much information as is available todetermine the composition of a library of candidate linker molecules.That library is then screened to identify a best linker.

In a third embodiment, if the folded structure of the protein is notknown or if the protein simply does not have a well-defined foldedstructure, then, using what knowledge about the protein does exist,simply select a linker to append the two PCCs. Even if an optimized,fully cooperative binder is not identified in this way, the linkedbiligand will almost certainly outperform either of the two monoligandsbecause of cooperativity effects.

In Vitro

For detection of IL-17F in solution, a capture agent of the inventioncan be detectably labeled, then contacted with the solution, andthereafter formation of a complex between the capture agent and theIL-17F target can be detected. As an example, a fluorescently labeledcapture agent can be used for in vitro IL-17F detection assays, whereinthe capture agent is added to a solution to be tested for IL-17F underconditions allowing binding to occur. The complex between thefluorescently labeled capture agent and the IL-17F target can bedetected and quantified by, for example, measuring the increasedfluorescence polarization arising from the complex-bound peptiderelative to that of the free peptide.

Alternatively, a sandwich-type “ELISA” assay can be used, wherein acapture agent is immobilized on a solid support such as a plastic tubeor well, then the solution suspected of containing IL-17F is contactedwith the immobilized binding moiety, non-binding materials are washedaway, and complexed polypeptide is detected using a suitable detectionreagent for recognizing IL-17F.

For detection or purification of soluble IL-17F from a solution, captureagents of the invention can be immobilized on a solid substrate such asa chromatographic support or other matrix material, then the immobilizedbinder can be loaded or contacted with the solution under conditionssuitable for formation of a capture agent/IL-17F complex. Thenon-binding portion of the solution can be removed and the complex canbe detected, for example, using an anti-IL-17F antibody, or ananti-binding polypeptide antibody, or the IL-17F can be released fromthe binding moiety at appropriate elution conditions.

In Vivo Diagnostic Imaging

A particularly preferred use for the capture agents of the invention isfor creating visually readable images of IL-17F or IL-17F-expressingcells in a biological fluid, such as, for example, in human serum. TheIL-17F capture agents disclosed herein can be converted to imagingreagents by conjugating the capture agents with a label appropriate fordiagnostic detection. Preferably, a capture agent exhibiting muchgreater specificity for IL-17F than for other serum proteins isconjugated or linked to a label appropriate for the detectionmethodology to be employed. For example, the capture agent can beconjugated with or without a linker to a paramagnetic chelate suitablefor Magnetic Resonance Imaging (MRI), with a radiolabel suitable forx-ray, Positron Emission Tomography (PET), Single Photon EmissionComputed Tomography (SPECT) or scintigraphic imaging (including achelator for a radioactive metal), with an ultrasound contrast agent(e.g., a stabilized microbubble, a microballoon, a microsphere or whathas been referred to as a gas filled “liposome”) suitable for ultrasounddetection, or with an optical imaging dye.

In another embodiment, rather than directly labeling a capture agentwith a detectable label or radiotherapeutic construct, one or morepeptides or constructs of the invention can be conjugated with forexample, avidin, biotin, or an antibody or antibody fragment that willbind the detectable label or radiotherapeutic.

A. Magnetic Resonance Imaging

The IL-17F capture agents described herein can advantageously beconjugated with a paramagnetic metal chelate in order to form a contrastagent for use in MRI.

Preferred paramagnetic metal ions have atomic numbers 21-29, 42, 44, or57-83. This includes ions of the transition metal or lanthanide serieswhich have one, and more preferably five or more, unpaired electrons anda magnetic moment of at least 1.7 Bohr magneton. Preferred paramagneticmetals include, but are not limited to, chromium (III), manganese (II),manganese (III), iron (II), iron (III), cobalt (II), nickel (II), copper(II), praseodymium (III), neodymium (III), samarium (III), gadolinium(III), terbium (III), dysprosium (III), holmium (III), erbium (III),europium (III) and ytterbium (III), chromium (III), iron (III), andgadolinium (III). The trivalent cation, Gd3+, is particularly preferredfor MRI contrast agents, due to its high relaxivity and low toxicity,with the further advantage that it exists in only one biologicallyaccessible oxidation state, which minimizes undesired metabolysis of themetal by a patient. Another useful metal is Cr3+, which is relativelyinexpensive. Gd(III) chelates have been used for clinical and radiologicMR applications since 1988, and approximately 30% of MRI exams currentlyemploy a gadolinium-based contrast agent.

The paramagnetic metal chelator is a molecule having one or more polargroups that act as a ligand for, and complex with, a paramagnetic metal.Suitable chelators are known in the art and include acids with methylenephosphonic acid groups, methylene carbohydroxamine acid groups,carboxyethylidene groups, or carboxymethylene groups. Examples ofchelators include, but are not limited to, diethylenetriaminepentaaceticacid (DTPA), 1,4,7,10-tetraazacyclo-tetradecane-1,4,7,10-tetraaceticacid (DOTA), 1-substituted1,4,7,-tricarboxymethyl-1,4,7,10-teraazacyclododecane (DO3A),ethylenediaminetetraacetic acid (EDTA), and1,4,8,11-tetra-azacyclotetradecane-1,4,8,11-tetraacetic acid (TETA).Additional chelating ligands are ethylene bis-(2-hydroxy-phenylglycine)(EHPG), and derivatives thereof, including 5-CI-EHPG, 5-Br-EHPG,5-Me-EHPG, 5-t-Bu-EHPG, and 5-sec-Bu-EHPG; benzodiethylenetriaminepentaacetic acid (benzo-DTPA) and derivatives thereof, includingdibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and dibenzylDTPA; bis-2 (hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) andderivatives thereof; the class of macrocyclic compounds which contain atleast 3 carbon atoms, more preferably at least 6, and at least twoheteroatoms (0 and/or N), which macrocyclic compounds can consist of onering, or two or three rings joined together at the hetero ring elements,e.g., benzo-DOTA, dibenzo-DOTA, and benzo-NOTA, where NOTA is1,4,7-triazacyclononane N,N′,N″-triacetic acid, benzo-TETA, benzo-DOTMA,where DOTMA is 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methyltetraacetic acid), and benzo-TETMA, where TETMA is1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic acid);derivatives of 1,3-propylene-diaminetetraacetic acid (PDTA) andtriethylenetetraaminehexaacetic acid (TTNA); derivatives of1,5,10-N,N′,N″-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM); and1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM). Apreferred chelator for use in the present invention is DTPA, and the useof DO3A is particularly preferred. Examples of representative chelatorsand chelating groups contemplated by the present invention are describedin WO 98/18496, WO 86/06605, WO 91/03200, WO 95/28179, WO 96/23526, WO97/36619, PCT/US98/01473, PCT/US98/20182, and U.S. Pat. Nos. 4,899,755,5,474,756, 5,846,519 and 6,143,274, all of which are hereby incorporatedby reference.

In accordance with the present invention, the chelator of the MRIcontrast agent is coupled to the IL-17F capture agent. The positioningof the chelate should be selected so as not to interfere with thebinding affinity or specificity of the IL-17F capture agent. The chelatealso can be attached anywhere on the capture agent.

In general, the IL-17F capture agent can be bound directly or covalentlyto the metal chelator (or other detectable label), or it can be coupledor conjugated to the metal chelator using a linker, which can be,without limitation, amide, urea, acetal, ketal, double ester, carbonyl,carbamate, thiourea, sulfone, thioester, ester, ether, disulfide,lactone, imine, phosphoryl, or phosphodiester linkages; substituted orunsubstituted saturated or unsaturated alkyl chains; linear, branched,or cyclic amino acid chains of a single amino acid or different aminoacids (e.g., extensions of the N- or C-terminus of the IL-17F bindingmoiety); derivatized or underivatized polyethylene glycols (PEGs),polyoxyethylene, or polyvinylpyridine chains; substituted orunsubstituted polyamide chains; derivatized or underivatized polyamine,polyester, polyethylenimine, polyacrylate, poly(vinyl alcohol),polyglycerol, or oligosaccharide (e.g., dextran) chains; alternatingblock copolymers; malonic, succinic, glutaric, adipic and pimelic acids;caproic acid; simple diamines and dialcohols; any of the other linkersdisclosed herein; or any other simple polymeric linkers known in the art(see, for example, WO 98/18497 and WO 98/18496). Preferably themolecular weight of the linker can be tightly controlled. The molecularweights can range in size from less than 100 to greater than 1000.Preferably the molecular weight of the linker is less than 100. Inaddition, it can be desirable to utilize a linker that is biodegradablein vivo to provide efficient routes of excretion for the imagingreagents of the present invention. Depending on their location withinthe linker, such biodegradable functionalities can include ester, doubleester, amide, phosphoester, ether, acetal, and ketal functionalities.

In general, known methods can be used to couple the metal chelate andthe IL-17F capture agent using such linkers (WO 95/28967, WO 98/18496,WO 98/18497 and discussion therein). The IL-17F binding moiety can belinked through an N- or C-terminus via an amide bond, for example, to ametal coordinating backbone nitrogen of a metal chelate or to an acetatearm of the metal chelate itself. The present disclosure contemplateslinking of the chelate on any position, provided the metal chelateretains the ability to bind the metal tightly in order to minimizetoxicity.

MRI contrast reagents prepared according to the disclosures herein canbe used in the same manner as conventional MRI contrast reagents.Certain MR techniques and pulse sequences can be preferred to enhancethe contrast of the site to the background blood and tissues. Thesetechniques include (but are not limited to), for example, black bloodangiography sequences that seek to make blood dark, such as fast spinecho sequences (Alexander, A. et al., 1998. Magn. Reson. Med., 40:298-310) and flow-spoiled gradient echo sequences (Edelman, R. et al.,1990. Radiology, 177: 45-50). These methods also include flowindependent techniques that enhance the difference in contrast, such asinversion-recovery prepared or saturation-recovery prepared sequencesthat will increase the contrast between IL-17F-expressing tissue andbackground tissues. Finally, magnetization transfer preparations alsocan improve contrast with these agents (Goodrich, K. et al., 1996.Invest. Radia, 31: 323-32).

The labeled reagent is administered to the patient in the form of aninjectable composition. The method of administering the MRI contrastagent is preferably parenterally, meaning intravenously,intraarterially, intrathecally, interstitially, or intracavitarilly. Forimaging IL-17F-expressing tissues, such as tumors, intravenous orintraarterial administration is preferred. For MRI, it is contemplatedthat the subject will receive a dosage of contrast agent sufficient toenhance the MR signal at the site IL-17F expression by at least 10%.After injection with the IL-17F capture agent containing MRI reagent,the patient is scanned in the MRI machine to determine the location ofany sites of IL-17F expression. In therapeutic settings, uponidentification of a site of IL-17F expression (e.g., fluid or tissue),an anti-cancer agent (e.g., inhibitors of IL-17F) can be immediatelyadministered, if necessary, and the patient can be subsequently scannedto visualize viral load.

B. Nuclear Imaging (Radionuclide Imaging) and Radiotherapy

The IL-17F capture agents of the invention can be conjugated with aradionuclide reporter appropriate for scintigraphy, SPECT, or PETimaging and/or with a radionuclide appropriate for radiotherapy.Constructs in which the IL-17F capture agents are conjugated with both achelator for a radionuclide useful for diagnostic imaging and a chelatoruseful for radiotherapy are within the scope of the invention.

For use as a PET agent a disclosed capture agent may be complexed withone of the various positron emitting metal ions, such as ⁵¹Mn, ⁵²Fe,⁶⁰Cu, ⁶⁸Ga, ⁷²As, ⁹⁴mTc, or ¹¹⁰In. The binding moieties of the inventioncan also be labeled by halogenation using radionuclides such as ¹⁸F,¹²⁴I, ¹²⁵I, ¹³¹I, ¹²³I, ⁷⁷Br, and ⁷⁶Br. Preferred metal radionuclidesfor scintigraphy or radiotherapy include ^(99m)Tc, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga,⁴⁷Sc, ⁵¹Cr, ¹⁶⁷Tm, ¹⁴¹Ce, ¹¹¹In, ¹⁶⁸Yb, ¹⁷⁵Yb, ¹⁴⁰La, ⁹⁰Y, ⁸⁸Y, ¹⁵³Sm,¹⁶⁶Ho, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶²Cu, ⁶⁴Cu, ⁹⁷Ru, ¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb,²¹¹Bi, ²¹²Bi, ²¹³Bi, ²¹⁴Bi, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹⁷mSn, ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁷⁷Lu,¹⁹⁸Au and ¹⁹⁹Au. The choice of metal will be determined based on thedesired therapeutic or diagnostic application. For example, fordiagnostic purposes the preferred radionuclides include ⁶⁴Cu, ⁶⁷Ga,⁶⁸Ga, ⁹⁹mTc, and ¹¹¹In. For therapeutic purposes, the preferredradionuclides include ⁶⁴Cu, ⁹⁰Y, ¹⁰⁵Rb, ¹¹¹In, ¹¹⁷mSn, ¹⁴⁹Pm, ¹⁵³Sm,¹⁶¹Tb, ¹⁶⁶Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb, ¹⁷⁷Ln, ^(186/188)Re, and ¹⁹⁹Au.^(99m)Tc is useful for diagnostic applications because of its low cost,availability, imaging properties, and high specific activity. Thenuclear and radioactive properties of 99mTc make this isotope an idealscintigraphic imaging agent. This isotope has a single photon energy of140 keV and a radioactive half-life of about 6 hours, and is readilyavailable from a ⁹⁹Mo-⁹⁹mTc generator. ¹⁸F, 4-[¹⁸F]fluorobenzaldehyde(¹⁸FB), Al[¹⁸F]-NOTA, ⁶⁸Ga-DOTA, and ⁶⁸Ga-NOTA are typical radionuclidesfor conjugation to IL-17F capture agents for diagnostic imaging.

The metal radionuclides may be chelated by, for example, linear,macrocyclic, terpyridine, and N₃S, N₂S₂, or N₄ chelants (see also, U.S.Pat. Nos. 5,367,080, 5,364,613, 5,021,556, 5,075,099, 5,886,142), andother chelators known in the art including, but not limited to, HYNIC,DTPA, EDTA, DOTA, DO3A, TETA, NOTA and bisamino bisthiol (BAT) chelators(see also U.S. Pat. No. 5,720,934). For example, N.sub.4 chelators aredescribed in U.S. Pat. Nos. 6,143,274; 6,093,382; 5,608,110; 5,665,329;5,656,254; and 5,688,487. Certain N.sub.35 chelators are described inPCT/CA94/00395, PCT/CA94/00479, PCT/CA95/00249 and in U.S. Pat. Nos.5,662,885; 5,976,495; and 5,780,006. The chelator also can includederivatives of the chelating ligandmercapto-acetyl-acetyl-glycyl-glycine (MAG3), which contains an N₃S, andN₂S₂ systems such as MAMA (monoamidemonoaminedithiols), DADS (N₂Sdiaminedithiols), CODADS and the like. These ligand systems and avariety of others are described in, for example, Liu, S, and Edwards,D., 1999. Chem. Rev., 99:2235-2268, and references therein.

The chelator also can include complexes containing ligand atoms that arenot donated to the metal in a tetradentate array. These include theboronic acid adducts of technetium and rhenium dioximes, such as aredescribed in U.S. Pat. Nos. 5,183,653; 5,387,409; and 5,118,797, thedisclosures of which are incorporated by reference herein, in theirentirety.

The chelators can be covalently linked directly to the IL-17F captureagent via a linker, as described previously, and then directly labeledwith the radioactive metal of choice (see, WO 98/52618, U.S. Pat. Nos.5,879,658, and 5,849,261).

IL-17F capture agents comprising ¹⁸F, 4-[¹⁸F]fluorobenzaldehyde (¹⁸FB),Al[¹⁸F]-NOTA, ⁶⁸Ga-DOTA, and ⁶⁸Ga-NOTA are of preferred interest fordiagnostic imaging. Complexes of radioactive technetium are also usefulfor diagnostic imaging, and complexes of radioactive rhenium areparticularly useful for radiotherapy. In forming a complex ofradioactive technetium with the reagents of this invention, thetechnetium complex, preferably a salt of ^(99m)Tc pertechnetate, isreacted with the reagent in the presence of a reducing agent. Preferredreducing agents are dithionite, stannous and ferrous ions; the mostpreferred reducing agent is stannous chloride. Means for preparing suchcomplexes are conveniently provided in a kit form comprising a sealedvial containing a predetermined quantity of a reagent of the inventionto be labeled and a sufficient amount of reducing agent to label thereagent with ^(99m)Tc. Alternatively, the complex can be formed byreacting a peptide of this invention conjugated with an appropriatechelator with a pre-formed labile complex of technetium and anothercompound known as a transfer ligand. This process is known as ligandexchange and is well known to those skilled in the art. The labilecomplex can be formed using such transfer ligands as tartrate, citrate,gluconate or mannitol, for example. Among the ^(99m)Tc pertechnetatesalts useful with the present invention are included the alkali metalsalts such as the sodium salt, or ammonium salts or lower alkyl ammoniumsalts.

Preparation of the complexes of the present invention where the metal isradioactive rhenium can be accomplished using rhenium starting materialsin the +5 or +7 oxidation state. Examples of compounds in which rheniumis in the Re(VII) state are NH₄ReO₄ or KReO₄. Re(V) is available as, forexample, [ReOCl₄](NBu₄), [ReOCl₄](AsPh₄), ReOCl₃(PPh₃)₂ and asReO₂(pyridine)⁴⁺, where Ph is phenyl and Bu is n-butyl. Other rheniumreagents capable of forming a rhenium complex also can be used.

Radioactively labeled PET, SPECT, or scintigraphic imaging agentsprovided by the present invention are encompassed having a suitableamount of radioactivity. Generally, the unit dose to be administered hasa radioactivity of about 0.01 mCi to about 100 mCi, preferably 1 mCi to20 mCi. The solution to be injected at unit dosage is from about 0.01 mLto about 10 mL. It is generally preferred to form radioactive complexesin solutions containing radioactivity at concentrations of from about0.01 mCi to 100 mCi per mL.

Typical doses of a radionuclide-labeled IL-17F capture agent accordingto the invention provide 10-20 mCi. After injection of theradionuclide-labeled IL-17F capture agents into the patient, a gammacamera calibrated for the gamma ray energy of the nuclide incorporatedin the imaging agent is used to image areas of uptake of the agent andquantify the amount of radioactivity present in the site. Imaging of thesite in vivo can take place in a matter of a few minutes. However,imaging can take place, if desired, in hours or even longer, after theradiolabeled peptide is injected into a patient. In most instances, asufficient amount of the administered dose will accumulate in the areato be imaged within about 0.1 of an hour to permit the taking ofscintiphotos.

Proper dose schedules for the radiotherapeutic compounds of the presentinvention are known to those skilled in the art. The compounds can beadministered using many methods including, but not limited to, a singleor multiple IV or IP injections, using a quantity of radioactivity thatis sufficient to cause damage or ablation of the targetedIL-17F-expressing tissue, but not so much that substantive damage iscaused to non-target (normal tissue). The quantity and dose required isdifferent for different constructs, depending on the energy andhalf-life of the isotope used, the degree of uptake and clearance of theagent from the body and the mass of the IL-17F-expressing tissue. Ingeneral, doses can range from a single dose of about 30-50 mCi to acumulative dose of up to about 3 Ci.

The radiotherapeutic compositions of the invention can includephysiologically acceptable buffers, and can require radiationstabilizers to prevent radiolytic damage to the compound prior toinjection. Radiation stabilizers are known to those skilled in the art,and can include, for example, para-aminobenzoic acid, ascorbic acid,gentistic acid and the like.

A single, or multi-vial kit that contains all of the components neededto prepare the complexes of this invention, other than the radionuclide,is an integral part of this invention.

A single-vial kit preferably contains a chelating ligand, a source ofstannous salt, or other pharmaceutically acceptable reducing agent, andis appropriately buffered with pharmaceutically acceptable acid or baseto adjust the pH to a value of about 3 to about 9. The quantity and typeof reducing agent used would depend on the nature of the exchangecomplex to be formed. The proper conditions are well known to those thatare skilled in the art. It is preferred that the kit contents be inlyophilized form. Such a single vial kit can optionally contain labileor exchange ligands such as glucoheptonate, gluconate, mannitol, malate,citric or tartaric acid and can also contain reaction modifiers such asdiethylenetriamine-pentaacetic acid (DPTA), ethylenediamine tetraaceticacid (EDTA), or alpha, beta, or gamma cyclodextrin that serve to improvethe radiochemical purity and stability of the final product. The kitalso can contain stabilizers, bulking agents such as mannitol, that aredesigned to aid in the freeze-drying process, and other additives knownto those skilled in the art.

A multi-vial kit preferably contains the same general components butemploys more than one vial in reconstituting the radiopharmaceutical.For example, one vial can contain all of the ingredients that arerequired to form a labile Tc(V) complex on addition of pertechnetate(e.g., the stannous source or other reducing agent). Pertechnetate isadded to this vial, and after waiting an appropriate period of time, thecontents of this vial are added to a second vial that contains theligand, as well as buffers appropriate to adjust the pH to its optimalvalue. After a reaction time of about 5 to 60 minutes, the complexes ofthe present invention are formed. It is advantageous that the contentsof both vials of this multi-vial kit be lyophilized. As above, reactionmodifiers, exchange ligands, stabilizers, bulking agents, etc. can bepresent in either or both vials.

Also provided herein is a method to incorporate an 18F radiolabeledprosthetic group onto an IL-17F capture agent. In one embodiment,4-[¹⁸F]fluorobenzaldehyde (¹⁸FB) is conjugated onto a capture agentbearing an aminooxy moiety, resulting in oxime formation. In anotherembodiment, [¹⁸F]fluorobenzaldehyde is conjugated onto a capture agentbearing an acyl hydrazide moiety, resulting in a hydrazone adduct.4-Fluorobenzaldehyde, can be prepared in ¹⁸F form by displacement of aleaving group, using ¹⁸F ion, by known methods.

¹⁸F-labeled capture agents can also be prepared from capture agentspossessing thiosemicarbazide moieties under conditions that promoteformation of a thiosemicarbozone, or by use of a ¹⁸F-labeled aldehydebisulfite addition complex.

The above methods are particularly amenable to the labeling of captureagents, e.g., the capture agents described herein, which can be modifiedduring synthesis to contain a nucleophilic hydroxylamine,thiosemicarbazide or hydrazine (or acyl hydrazide) moiety that can beused to react with the labeled aldehyde. The methods can be used for anycapture agent that can accommodate a suitable nucleophilic moiety.Typically the nucleophilic moiety is appended to the N-terminus of thepeptide, but the skilled artisan will recognize that the nucleophilealso can be linked to an amino acid side chain or to the peptideC-terminus. Methods of synthesizing a radiolabeled peptide sequence areprovided in which 4-[¹⁸F]fluorobenzaldehyde is reacted with a peptidesequence comprising either a hydroxylamine, a thiosemicarbazide or ahydrazine (or acyl hydrazide) group, thereby forming the correspondingoximes, thiosemicarbazones or hydrazones, respectively. The4-[¹⁸F]fluorobenzaldehyde typically is generated in situ by theacid-catalyzed decomposition of the addition complex of4-[¹⁸F]fluorobenzaldehyde and sodium bisulfite. The use of the bisulfiteaddition complex enhances the speed of purification since, unlike thealdehyde, the complex can be concentrated to dryness. Formation of thecomplex is also reversible under acidic and basic conditions. Inparticular, when the complex is contacted with a peptide containing ahydroxylamine, a thiosemicarbazide or a hydrazine (or acyl hydrazide)group in acidic medium, the reactive free 4-[¹⁸F]fluorobenzaldehyde isconsumed as it is formed in situ, resulting in the corresponding ¹⁸Fradiolabeled peptide sequence.

In the instances when the oxime, thiosemicarbazone or hydrazone linkagespresent in vivo instability, an additional reduction step may beemployed to reduce the double bond connecting the peptide to the ¹⁸Fbearing substrate. The corresponding reduced peptide linkage wouldenhance the stability. One of skill in the art would appreciate thevariety of methods available to carry out such a reduction step.Reductive amination steps as described in Wilson et al., Journal ofLabeled Compounds and Radiopharmaceuticals, XXVIII (10), 1189-1199, 1990may also be used to form a Schiffs base involving a peptide and4-[¹⁸F]fluorobenzaldehyde and directly reducing the Schiffs base usingreducing agents such as sodium cyanoborohydride.

The 4-[¹⁸F]fluorobenzaldehyde may be prepared as described in Wilson etal., Journal of Labeled Compounds and Radiopharmaceuticals, XXVIII (10),1189-1199, 1990; Iwata et al., Applied radiation and isotopes, 52,87-92, 2000; Poethko et al., The Journal of Nuclear Medicine, 45,892-902, 2004; and Schottelius et al., Clinical Cancer Research, 10,3593-3606, 2004. The Na18F in water may be added to a mixture ofkryptofix and K.sub.2CO.sub.3. Anhydrous acetonitrile may be added andthe solution is evaporated in a heating block under a stream of argon.Additional portions of acetonitrile may be added and evaporated tocompletely dry the sample. The 4-trimethylammoniumbenzaldehyde triflatemay be dissolved in DMSO and added to the dried F-18. The solution maythen be heated in the heating block. The solution may be cooled briefly,diluted with water and filtered through a Waters®. Oasis HLB LPextraction cartridge. The cartridge may be washed with 9:1water:acetonitrile and water to remove unbound ¹⁸F and unreacted4-trimethylammoniumbenzaldehyde triflate. The 4-[¹⁸F]fluorobenzaldehydemay then be eluted from the cartridge with methanol in fractions.

Therapeutic Applications

Provided herein in certain embodiments are methods of using the IL-17Fcapture agents disclosed herein to identify, detect, quantify, and/orseparate IL-17F in a biological sample. In certain embodiments, thesemethods utilize an immunoassay, with the capture agent replacing anantibody or its equivalent. In certain embodiments, the immunoassay maybe a Western blot, pull-down assay, dot blot, or ELISA.

A biological sample for use in the methods provided herein may beselected from the group consisting of organs, tissue, bodily fluids, andcells. Where the biological sample is a bodily fluid, the fluid may beselected from the group consisting of blood, serum, plasma, urine,sputum, saliva, stool, spinal fluid, cerebral spinal fluid, lymph fluid,skin secretions, respiratory secretions, intestinal secretions,genitourinary tract secretions, tears, and milk. The organs include,e.g., the adrenal glands, bladder, bones, brain, breasts, cervix,esophagus, eyes, gall bladder, genitals, heart, kidneys, largeintestine, liver, lungs, lymph nodes, ovaries, pancreas, pituitarygland, prostate, salivary glands, skeletal muscles, skin, smallintestine, spinal cord, spleen, stomach, thymus gland, trachea, thyroid,testes, ureters, and urethra. Tissues include, e.g., epithelial,connective, nervous, and muscle tissues.

Provided herein in certain embodiments are methods of using the IL-17Fcapture agents disclosed herein to diagnose and/or classify (e.g.,stage) a condition associated with IL-17F expression. In certain ofthese embodiments, the methods comprise (a) obtaining a biologicalsample from a subject; (b) measuring the presence or absence of IL-17Fin the sample with the IL-17F capture agent; (c) comparing the levels ofIL-17F to a predetermined control range for IL-17F; and (d) diagnosing acondition associated with IL-17F expression based on the differencebetween IL-17F levels in the biological sample and the predeterminedcontrol.

In other embodiments, the IL-17F capture agents disclosed herein areused as a mutant specific targeted therapeutic. In certain aspects ofthis embodiment, the IL-17F capture agent is administered alone withoutdelivering DNA, a radiopharmaceutical or another active agent.

The IL-17F capture agents of the invention also can be used to targetgenetic material to IL-17F expressing cells. The genetic material caninclude nucleic acids, such as RNA or DNA, of either natural orsynthetic origin, including recombinant RNA and DNA and antisense RNAand DNA. Types of genetic material that can be used include, forexample, genes carried on expression vectors such as plasmids,phagemids, cosmids, yeast artificial chromosomes (YACs) and defective or“helper” viruses, antigene nucleic acids, both single and doublestranded RNA and DNA and analogs thereof, such as phosphorothioate andphosphorodithioate oligodeoxynucleotides. Additionally, the geneticmaterial can be combined, for example, with lipids, proteins or otherpolymers. Delivery vehicles for genetic material can include, forexample, a virus particle, a retroviral or other gene therapy vector, aliposome, a complex of lipids (especially cationic lipids) and geneticmaterial, a complex of dextran derivatives and genetic material, etc. Inan embodiment the capture agents of the invention are utilized in genetherapy.

In this embodiment, genetic material, or one or more delivery vehiclescontaining genetic material can be conjugated to one or more IL-17Fcapture agents of this disclosure and administered to a patient.

Therapeutic agents and the IL-17F capture agents disclosed herein can belinked or fused in known ways, optionally using the same type of linkersdiscussed elsewhere in this application. Preferred linkers will besubstituted or unsubstituted alkyl chains, amino acid chains,polyethylene glycol chains, and other simple polymeric linkers known inthe art. More preferably, if the therapeutic agent is itself a protein,for which the encoding DNA sequence is known, the therapeutic proteinand IL-17F binding polypeptide can be coexpressed from the samesynthetic gene, created using recombinant DNA techniques, as describedabove. The coding sequence for the IL-17F binding polypeptide can befused in frame with that of the therapeutic protein, such that thepeptide is expressed at the amino- or carboxy-terminus of thetherapeutic protein, or at a place between the termini, if it isdetermined that such placement would not destroy the required biologicalfunction of either the therapeutic protein or the IL-17F bindingpolypeptide. A particular advantage of this general approach is thatconcatamerization of multiple, tandemly arranged IL-17F capture agentsis possible, thereby increasing the number and concentration of IL-17Fbinding sites associated with each therapeutic protein. In this manner,IL-17F binding avidity is increased, which would be expected to improvethe efficacy of the recombinant therapeutic fusion protein.

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

EXAMPLES Example 1. IL-17F Epitope Design

The primary sequences of IL-17F and IL-17A were examined to understandwhere there are regions of identity or similarity, and where there aredifferences. To create macrocyclic peptide ligands that can effectivelydiscriminate IL-17F from IL-17A, attention was placed on those sequencesthat are unique to the two proteins. Sequence differences thatdiscriminate IL-17F from IL-17A were found to occur in the N-terminalregion of the mature proteins. In IL-17F, this region of uniquenesscorresponds to Arg-31 to Thr-79 (FIG. 1A).

Two polypeptide epitopes were chemically synthesized and exploited asthe targets for generating specific macrocyclic peptide ligands againstIL-17F. Epitopes were designed with a biotin-PEG₃ assay handle and astrategically substituted azide click handle (Az4=L-azidolysine). Thesite of click handle substitution in each epitope was determined byexamining the protein structure and identifying an amino acid (a) whoseside chain is surface-exposed and does not interact with other atoms inthe protein, and (b) that is chemically similar to Az4. IL-17F Epitope1(Phe-40 to Ser-54) is located very close to the N-terminus of IL-17F,and is substituted with Az4 at a central location (Cys-48). IL-17FEpitope2 (Gly-60 to Ser-69) is found after Epitope1 in the primarysequence, and is substituted with Az4 at Ile-62. The sequences ofEpitope1 and Epitope2 are shown in FIGS. 1B and C.

Example 2. Screening for Macrocycle Anchors Against IL-17F Epitope1

Screens were performed using a triazole-cyclized OBOC library of theform H₂N-Pra-Cy(XXXXX)-Met-TG (SEQ ID NO:34), where TG=TentaGel® S NH₂resin (S 30 902, Rapp Polymere), X=one of seventeen L-amino acids(lacking Cys, Met, and Ile), Pra=L-propargylglycine, and Cy( )=triazolecyclization via flanking Pra and Az4 residues. Macrocycles wereidentified against the IL-17F Epitope1 fragment using a four-stepscreening process: 1) a pre-clear to eliminate non-specific binders, 2)a product screen to identify hits resulting from epitope-templated insitu click chemistry, 3) a target screen against His-tagged IL-17Fprotein, and 4) another target screen against His-tagged IL-17F proteinin 2% (v/v) human serum to identify peptides whose binding to IL-17F isunperturbed by serum proteins.

Pre-clear. Swelled library beads (500 mg) were blocked overnight withBlocking Buffer (25 mM Tris-HCl, 150 mM NaCl, 1% (w/v) BSA, and 0.05%(v/v) Tween-20, pH 7.6) at 4° C., then washed with Blocking Buffer threetimes. A 1:10,000 dilution of Streptavidin-Alkaline Phosphatase (V559C,Promega) in 5 mL Blocking Buffer was added to the beads and incubatedwith gentle shaking at room temperature for 1 h. The beads weresubsequently washed with 3×3 mL TBS (25 mM Tris-HCl, 150 mM NaCl, pH7.6) (1 min ea), 3×3 mL 0.1 M glycine pH 2.8 wash buffer, 3×3 mL TBS,then 3×3 mL Alkaline Phosphatase buffer (100 mM Tris-HCl, 150 mM NaCl, 1mM MgCl₂, pH 9) (5 min ea). Binding was visualized by incubating thebeads in the presence of 5-bromo-4-chloro-3-indolyl phosphate/nitro bluetetrazolium (BCIP/NBT) substrate (S3771, Promega) for 25 min. Purplebeads indicated background binders and were removed by pipet anddiscarded. The remaining clear beads were collected and stripped with7.5 M guanidine hydrochloride pH 2.0 for 30 min, washed ten times withwater, and incubated in 1-methyl-2-pyrrolidinone (NMP) overnight todecolorize.

Product Screen with IL-17F Epitope1. Beads remaining from the pre-clearwere washed with water ten times and TBS three times. Beads were thenincubated with 3 mL of 100 μM IL-17F Epitope1 fragment(Biotin-PEG₃-FFQKPES(SEQ ID NO:1)[Az4]PPVPGGS(SEQ ID NO:32)) in TBS for1.5 h at room temperature to allow for an in situ click reaction tooccur. The beads were washed with TBS ten times and then incubated with7.5 M guanidine hydrochloride pH 2.0 for 1 h to remove all IL-17Fepitope not attached covalently to the beads. These beads were washedwith TBS ten times and re-blocked with Blocking Buffer for 2 h. A1:10,000 dilution of Streptavidin-Alkaline Phosphatase in 5 mL BlockingBuffer was added for 1 h to detect the presence of IL-17F epitopeclicked to beads. The beads were subsequently washed with 3×3 mL TBS (1min ea), 3×3 mL 0.1 M glycine pH 2.8 wash buffer, 3×3 mL TBS, then 3×3mL Alkaline Phosphatase (pH 9) buffer (5 min ea). After this, the beadswere developed with BCIP/NBT for 25 min as outlined in the pre-clear.Purple epitope-conjugated hit beads were selected by pipet and saved.These hits (25 total: 5 dark purple, 20 medium to light purple) weretreated with 7.5 M guanidine hydrochloride pH 2.0 for 30 min to removeattached streptavidin, washed ten times with water, and incubated in NMPovernight to decolorize.

Target Screen with His-tagged IL-17F Protein. Product hits were washedwith water ten times and stored in TBS at 4° C. These 25 beads weretransferred to a Corning® 8162 Costar® Spin-X® centrifuge tube filter(cellulose acetate membrane) and incubated with Blocking Buffer for 3 hat room temperature. The beads were rinsed three times with BlockingBuffer and then incubated with 150 nM of full-length His-tagged IL-17Fprotein (ab167911, Abcam) in Blocking Buffer (preparation: 0.5 μLHis-tagged IL-17F protein in 200 μL Blocking Buffer) for 1 h at roomtemperature. The beads were washed three times with Blocking Buffer andthen incubated with 500 μL of 1:10,000 Anti-6× His Tag® antibody [HIS-1](Alkaline Phosphatase-conjugated) (ab49746, Abcam) in Blocking Bufferfor 1 h at room temperature. The beads were subsequently washed with3×500 μL Blocking Buffer, 3×500 μL TBS, then 3×500 μL AlkalinePhosphatase (pH 9) buffer (centrifuging at 7000 rpm for 30 sec aftereach wash). After this, the beads were developed with BCIP/NBT for 10min. Purple hit beads bound to IL-17F protein were selected by pipet andsaved. 20 beads were purple indicating binding to both the IL-17 epitopeand protein, while 5 were clear indicating no binding to IL-17F protein.The 20 target hits were treated with 7.5 M guanidine hydrochloride pH2.0 for 30 min to remove bound proteins, washed ten times with water,and incubated in NMP overnight to decolorize. Target Screen withHis-tagged IL-17F Protein in 2% (v/v) Human Serum.

Target hits were washed with water ten times. These 20 beads wereincubated with Blocking Buffer for 7 h in a Corning® 8162 Costar®Spin-X® centrifuge tube filter (cellulose acetate membrane). The beadswere rinsed three times with Blocking Buffer and then incubated with 150nM of full-length His-tagged IL-17F protein (ab167911, Abcam) inBlocking Buffer containing 2% (v/v) human serum (HS-30, OmegaScientific) for 1 h at room temperature (preparation: 1.25 μL His-taggedIL-17F protein+10 μL filtered serum+490 μL Blocking Buffer). Note:Before the screen, particulate matter was removed from serum bycentrifugation (7000 rpm, 30 sec) using a Corning® 8162 Costar® Spin-X®tube filter. The beads were washed three times with Blocking Buffer andthen incubated with 500 μL of 1:10,000 Anti-6× His Tag® antibody [HIS-1](Alkaline Phosphatase-conjugated) (ab49746, Abcam) in Blocking Bufferfor 1 h at room temperature. The beads were subsequently washed with3×500 μL Blocking Buffer, 3×500 μL TBS, then 3×500 μL AlkalinePhosphatase (pH 9) buffer (centrifuging at 7000 rpm for 30 sec aftereach wash). After this, the beads were developed with BCIP/NBT for 10min. Purple hit beads were selected by pipet and saved. The 2 hits whosebinding to IL-17F protein was unperturbed by serum proteins were treatedwith 7.5 M guanidine hydrochloride pH 2.0 for 30 min to remove boundproteins, washed ten times with water, and incubated in NMP overnight todecolorize. The 2 hits were finally washed with water ten times toprepare for sequencing analysis.

Sequencing was performed via Edman degradation on an Applied BiosystemsProcise® cLC 2-cartridge system in the Protein/Peptide Micro AnalyticalLaboratory at Caltech. The Edman sequencer was unable to distinguishbetween 1) residues K (lysine) and L (leucine), and 2) residues Q(glutamine) and T (threonine). Sequencing results are shown in Table 1including the K/L and Q/T variants.

TABLE 1 Sequences of macrocyclic peptide hits identified against IL-17FEpitope1 x2 x3 x4 x5 x6 hit1 F Y K T H F Y K Q H F Y L T H F Y L Q Hhit2 R R A T S R R A Q S

These candidate peptides were re-synthesized on a cleavable resin,purified by reversed phase HPLC using a C18 column (Phenomenex Luna, 5μm, 250×10 mm), and tested for affinity and specificity against theIL-17F protein by ELISA.

Synthesis Data for IL-17F Epitope1 Hits

Cy(FYKTH)(SEQ ID NO:5)-PEG₃-biotin. MALDI-MS (m/z): calcd. forC₆₅H₉₇N₁₇O₁₄S (M+H) 1372.71; found 1374.24.

Cy(FYKQH)(SEQ ID NO:6)-PEG₃-biotin. MALDI-MS (m/z): calcd. forC₆₆H₉₈N₁₈O₁₄S (M+H) 1399.72; found 1401.36.

Cy(FYLTH)(SEQ ID NO:7)-PEG₃-biotin. MALDI-MS (m/z): calcd. forC₆₅H₉₆N₁₆O₁₄S (M+H) 1357.70; found 1360.15.

Cy(FYLQH)(SEQ ID NO:8)-PEG₃-biotin. MALDI-MS (m/z): calcd. forC₆₆H₉₇N₁₇O₁₄S (M+H) 1384.71; found 1386.24.

Cy(RRATS)(SEQ ID NO:9)-PEG₃-biotin. MALDI-MS (m/z): calcd. forC₅₃H₉₄N₂₀O₁₄S (M+H) 1269.70; found 1269.91 (FIG. 2C).

Cy(RRAQS)(SEQ ID NO:10)-PEG₃-biotin. MALDI-MS (m/z): calcd. forC₅₄H₉₅N₂₁O₁₄S (M+H) 1295.71; found 1296.19 (FIG. 2D).

Example 3. In Vitro Assays with IL-17F Epitope1 Targeted Ligands

Sandwich ELISA. A black 96-well NeutrAvidin Coated High Binding Capacityplate (15510, Pierce) was coated with 2 μM macrocyclic peptide ligand inTBS (25 mM Tris-HCl, 150 mM NaCl, pH 7.6) for 2 h at room temperature.Biotinylated monoclonal anti-IL17F (TA319597, Origene) was coated at 4μg/mL in TBS as a control. The plate was aspirated and then washed withTBS (5×) and Wash Buffer (0.05% (v/v) Tween-20 in PBS, 1×). Full-lengthHis-tagged IL-17F protein (ab167911, Abcam) was serially diluted in WashBuffer (from 800 to 0 nM) and incubated in the designated microwells for90 min at room temperature. Microwells were aspirated and subsequentlywashed with Wash Buffer (10×). To detect the bound IL-17F protein,Alkaline Phosphatase (AP)-conjugated Anti-6×His Tag® antibody [HIS-1](ab49746, Abcam) was prepared at 1:10,000 dilution and added to themicrowells for 1 h at room temperature. The plate was aspirated andwashed with Wash Buffer (5×). AttoPhos® AP Fluorescent Substrate System(S1000, Promega) was employed to develop the microwells. Using anexcitation wavelength of 430 nm, fluorescent emission at 535 nm wasrecorded by Beckman Coulter DTX880 photometer. Titration curves were fitusing a four-parameter regression curve fitting program (Origin 8.5) todetermine EC₅₀ values.

Results are shown in FIG. 2A. The binding affinity ofPEG₃-biotin-modified Cy(RRATS) (SEQ ID NO:9) and Cy(RRAQS) (SEQ IDNO:10) was tested in an ELISA format. For these assays, a dilutionseries of full-length His-tagged IL-17F protein was captured using themacrocyclic peptide ligands immobilized on a NeutrAvidin-coated plate.Cy (RRATS) (SEQ ID NO:9) and Cy(RRAQS) (SEQ ID NO:10) exhibited EC₅₀values of 66±9 nM and 52±5 nM, respectively, for human IL-17F protein. Asimilarly assayed biotinylated monoclonal anti-IL17F shows similarbinding affinity.

Point ELISA (IL-17F vs. IL-17A selectivity assay). A black 96-wellNeutrAvidin Coated High Binding Capacity plate (15510, Pierce) wascoated with 2 μM macrocyclic peptide ligand in TBS (pH 7.6) for 2 h atroom temperature. The plate was aspirated and then washed with TBS (5×)and Wash Buffer (0.05% (v/v) Tween-20 in PBS, 1×). Full-lengthHis-tagged IL-17F (ab167911, Abcam) and IL-17A (ab166882, Abcam)proteins were prepared at 200 and 50 nM in Wash Buffer and incubated inthe designated microwells for 90 min at room temperature. Microwellswere aspirated and subsequently washed with Wash Buffer (10×). To detectthe bound IL-17F and IL-17A proteins, Alkaline Phosphatase(AP)-conjugated Anti-6×His Tag® antibody [HIS-1] (ab49746, Abcam) wasprepared at 1:10,000 dilution and added to the microwells for 1 h atroom temperature. The plate was aspirated and washed with Wash Buffer(5×). AttoPhos® AP Fluorescent Substrate System (S1000, Promega) wasemployed to develop the microwells. Using an excitation wavelength of430 nm, fluorescent emission at 535 nm was recorded by Beckman CoulterDTX880 photometer.

Results are shown in FIG. 2B. The selectivity of PEG₃-biotin-modifiedCy(RRATS) (SEQ ID NO:9) and Cy(RRAQS) (SEQ ID NO:10) was tested in anELISA format. For these assays, the full-length His-tagged IL-17F andIL-17A proteins were captured using the macrocyclic peptide ligandsimmobilized on a NeutrAvidin-coated plate. Both Cy(RRATS) (SEQ ID NO:9)and Cy(RRAQS) (SEQ ID NO:10) exhibited 2:1 selectivity for IL-17F overthe 200-50 nM concentration range. These results confirm the selectivenature of the epitope-targeting strategy.

Assay to determine orientation of macrocycle binding to IL-17F Epitope1.A black 96-well NeutrAvidin Coated High Binding Capacity plate (15510,Pierce) was coated with 2 μM macrocyclic peptide ligand in TBS (pH 7.6)for 2 h at room temperature. Biotinylated monoclonal anti-IL17F(TA319597, Origene) was coated at 4 μg/mL in TBS as a control. The platewas aspirated and then washed with TBS (5×) and Wash Buffer (0.05% (v/v)Tween-20 in PBS, 1×). Chemically synthesized His-tagged IL-17F epitopeswere prepared at 2 μM in Wash Buffer and incubated in the designatedmicrowells for 90 min at room temperature. Wash Buffer without epitopewas added as a control. Microwells were aspirated and subsequentlywashed with Wash Buffer (10×). To detect the bound IL-17F epitopes,Alkaline Phosphatase (AP)-conjugated Anti-6×His Tag® antibody [HIS-1](ab49746, Abcam) was prepared at 1:10,000 dilution and added to themicrowells for 1 h at room temperature. The plate was aspirated andwashed with Wash Buffer (5×). AttoPhos® AP Fluorescent Substrate System(S1000, Promega) was employed to develop the microwells. Using anexcitation wavelength of 430 nm, fluorescent emission at 535 nm wasrecorded by Beckman Coulter DTX880 photometer. Data are shown aftersubtraction of the no-epitope background.

Results are shown in FIG. 3. For this experiment, the IL-17F Epitope1was re-synthesized with a His₆ assay handle and C48S substitutioninstead of a click handle. Two additional His-tagged IL-17F epitopeswere synthesized to contain strategic scrambling of the sequences eitherN-terminal or C-terminal to C48S. Point ELISAs for the three His-taggedIL-17F epitopes were conducted against the immobilized macrocyclicpeptide ligands. For PEG₃-biotin-modified Cy(RRATS) (SEQ ID NO:9) andCy(RRAQS) (SEQ ID NO:10), positive ELISA signals were obtained for theHis-tagged IL-17F Epitope1 (C48S) and the epitope scrambled C-terminalto click handle (black and red bars). Binding was destroyed when theepitope was scrambled N-terminal to the click handle (blue bars).

Therefore, the macrocyclic peptide ligands show preferential binding tothe sequence FFQKPES (SEQ ID NO:1) within IL-17F Epitope1 (C48S). On theother hand, biotinylated monoclonal anti-IL17F showed no appreciablebinding to the His-tagged IL-17F epitopes as it was raised against aprotein immunogen.

In summary, the results of FIGS. 2-3 confirm that the epitope-targetingstrategy produced sub-100 nM affinity macrocyclic peptide ligands thatselectively recognize IL-17F epitopes as well as the full-lengthprotein.

Example 4. Screening for Macrocycle Anchor Against IL-17F Epitope2

Screens were similarly performed to target the IL-17F Epitope2 fragment(Biotin-PEG₃-GI[Az4]NENQRVS) (SEQ ID NO:33) using the triazole-cyclizedOBOC library of the form H₂N-Pra-Cy(XXXXX)-Met-TG (SEQ ID NO:34).Macrocycles were identified against the IL-17F Epitope2 fragment usingthe aforementioned four-step screening process: 1) a pre-clear toeliminate non-specific binders, 2) a product screen to identify hitsresulting from epitope-templated in situ click chemistry, 3) a targetscreen against His-tagged IL-17F protein, and 4) additional targetscreens against His-tagged IL-17F protein in 1% to 5% (v/v) human serumto identify peptides whose binding to IL-17F is unperturbed by serumproteins.

Product Screen with IL-17F Epitope2. The product screen was conductedwith 3 mL of 100 μM IL-17F Epitope2 fragment(Biotin-PEG₃-GI[Az4]NENQRVS) (SEQ ID NO:33) in TBS for 1.5 h at roomtemperature to allow for an in situ click reaction to occur. The beadswere washed with TBS ten times and then incubated with 7.5 M guanidinehydrochloride pH 2.0 for 1 h to remove all IL-17F epitope not attachedcovalently to the beads. These beads were washed with TBS ten times andre-blocked with Blocking Buffer for 2 h. A 1:10,000 dilution ofStreptavidin-Alkaline Phosphatase in 5 mL Blocking Buffer was added for1 h to detect the presence of IL-17F epitope clicked to beads. The beadswere subsequently washed with 3×3 mL TBS (1 min ea), 3×3 mL 0.1 Mglycine pH 2.8 wash buffer, 3×3 mL TBS, then 3×3 mL Alkaline Phosphatase(pH 9) buffer (5 min ea). After this, the beads were developed withBCIP/NBT for 25 min as outlined in the pre-clear. Purpleepitope-conjugated hit beads were selected by pipet and saved. Thesehits (98 total: 50 dark purple, 48 medium to light purple) were treatedwith 7.5 M guanidine hydrochloride pH 2.0 for 30 min to remove attachedstreptavidin, washed ten times with water, and incubated in NMPovernight to decolorize.

Target Screen with His-tagged IL-17F Protein. Product hits were washedwith water ten times and stored in TBS at 4° C. These 98 beads weretransferred to a Corning® 8162 Costar® Spin-X® centrifuge tube filter(cellulose acetate membrane) and incubated with Blocking Buffer for 3 hat room temperature. The beads were rinsed three times with BlockingBuffer and then incubated with 150 nM of full-length His-tagged IL-17Fprotein (ab167911, Abcam) in Blocking Buffer (preparation: 0.5 μLHis-tagged IL-17F protein in 200 μL Blocking Buffer) for 1 h at roomtemperature. The beads were washed three times with Blocking Buffer andthen incubated with 500 μL of 1:10,000 Anti-6×His Tag® antibody [HIS-1](Alkaline Phosphatase-conjugated) (ab49746, Abcam) in Blocking Bufferfor 1 h at room temperature. The beads were subsequently washed with3×500 μL Blocking Buffer, 3×500 μL TBS, then 3×500 μL AlkalinePhosphatase (pH 9) buffer (centrifuging at 7000 rpm for 30 sec aftereach wash). After this, the beads were developed with BCIP/NBT for 10min. Purple hit beads bound to IL-17F protein were selected by pipet andsaved. 53 beads were purple indicating binding to both the IL-17 epitopeand protein, while 40 were clear indicating no binding to IL-17Fprotein. The 53 target hits were treated with 7.5 M guanidinehydrochloride pH 2.0 for 30 min to remove bound proteins, washed tentimes with water, and incubated in NMP overnight to decolorize.

Target Screen with His-tagged IL-17F Protein in 1% (v/v) Human Serum.Target hits were washed with water ten times. These 53 beads wereincubated with Blocking Buffer for 7 h in a Corning® 8162 Costar®Spin-X® centrifuge tube filter (cellulose acetate membrane). The beadswere rinsed three times with Blocking Buffer and then incubated with 150nM of full-length His-tagged IL-17F protein (ab167911, Abcam) inBlocking Buffer containing 1% (v/v) human serum (HS-30, OmegaScientific) for 1 h at room temperature (preparation: 1.25 μL His-taggedIL-17F protein+5 μL filtered serum+495 μL Blocking Buffer). Note: Beforethe screen, particulate matter was removed from serum by centrifugation(7000 rpm, 30 sec) using a Corning® 8162 Costar® Spin-X® tube filter.The beads were washed three times with Blocking Buffer and thenincubated with 500 μL of 1:10,000 Anti-6×His Tag® antibody [HIS-1](Alkaline Phosphatase-conjugated) (ab49746, Abcam) in Blocking Bufferfor 1 h at room temperature. The beads were subsequently washed with3×500 μL Blocking Buffer, 3×500 μL TBS, then 3×500 μL AlkalinePhosphatase (pH 9) buffer (centrifuging at 7000 rpm for 30 sec aftereach wash). After this, the beads were developed with BCIP/NBT for 10min. Purple hit beads were selected by pipet and saved. The 23 hitswhose binding to IL-17F protein was unperturbed by serum proteins weretreated with 7.5 M guanidine hydrochloride pH 2.0 for 30 min to removebound proteins, washed ten times with water, and incubated in NMPovernight to decolorize.

Target Screen with His-tagged IL-17F Protein in 5% (v/v) Human Serum toRefine the Number of Hits. The 23 beads were washed with water ten timesand then incubated with Blocking Buffer for 7 h in a Corning® 8162Costar® Spin-X® centrifuge tube filter (cellulose acetate membrane). Thebeads were rinsed three times with Blocking Buffer and then incubatedwith 150 nM of full-length His-tagged IL-17F protein (ab167911, Abcam)in Blocking Buffer containing 5% (v/v) human serum (HS-30, OmegaScientific) for 1 h at room temperature (preparation: 1.25 μL His-taggedIL-17F protein+25 μL filtered serum+475 μL Blocking Buffer). Note:Before the screen, particulate matter was removed from serum bycentrifugation (7000 rpm, 30 sec) using a Corning® 8162 Costar® Spin-X®tube filter. The beads were washed three times with Blocking Buffer andthen incubated with 500 μL of 1:10,000 Anti-6×His Tag® antibody [HIS-1](Alkaline Phosphatase-conjugated) (ab49746, Abcam) in Blocking Bufferfor 1 h at room temperature. The beads were subsequently washed with3×500 μL Blocking Buffer, 3×500 μL TBS, then 3×500 μL AlkalinePhosphatase (pH 9) buffer (centrifuging at 7000 rpm for 30 sec aftereach wash). After this, the beads were developed with BCIP/NBT for 10min. Purple hit beads were selected by pipet and saved. The 6 hits whosebinding to IL-17F protein was unperturbed by the increased background ofserum proteins were treated with 7.5 M guanidine hydrochloride pH 2.0for 30 min to remove bound proteins, washed ten times with water, andincubated in NMP overnight to decolorize. The 6 hits were finally washedwith water ten times to prepare for sequencing analysis.

Sequencing was performed via Edman degradation. The Edman sequencer wasunable to distinguish between 1) residues K (lysine) and L (leucine),and 2) residues Q (glutamine) and T (threonine). Sequencing results areshown in Table 2 including the K/L and Q/T variants.

TABLE 2 Sequences of macrocyclic peptide hits identified against IL-17FEpitope2 x2 x3 x4 x5 x6 hit1 K Y G E V L Y G E V hit2 V H K S G V H L SG hit3 Q K H G P T K H G P Q L H G P T L H G P hit4 Y D L Q R Y D L T RY D K Q R Y D K T R hit5 K K G W P K L G W P L K G W P L L G W P hit6 RS Y N L R S Y N K

These candidate peptides were re-synthesized on a cleavable resin,purified by reversed phase HPLC using a C18 column, and tested againstthe IL-17F protein by ELISA.

Synthesis Data for IL-17F Epitope2 Hits

Cy(KYGEV)(SEQ ID NO:11)-PEG₃-biotin. MALDI-MS (m/z): calcd. forC₅₈H₉₃N₁₅O₁₅S (M+H) 1272.67; found 1274.02.

Cy(LYGEV)(SEQ ID NO:12)-PEG₃-biotin. MALDI-MS (m/z): calcd. forC₅₈H₉₂N₁₄O₁₅S (M+H) 1257.66; found 1259.13.

Cy(VHKSG)(SEQ ID NO:13)-PEG₃-biotin. MALDI-MS (m/z): calcd. forC₅₃H₈₉N₁₇O₁₃S (M+H) 1204.65; found 1206.20.

Cy(VHLSG)(SEQ ID NO:14)-PEG₃-biotin. MALDI-MS (m/z): calcd. forC₅₃H₈₈N₁₆O₁₃S (M+H) 1189.64; found 1191.11.

Cy(QKHGP)(SEQ ID NO:15)-PEG₃-biotin. MALDI-MS (m/z): calcd. forC₅₅H₉₀N₁₈O₁₃S (M+H) 1243.67; found 1245.18.

Cy(TKHGP)(SEQ ID NO:16)-PEG₃-biotin. MALDI-MS (m/z): calcd. forC₅₄H₈₉N₁₇O₁₃S (M+H) 1216.65: found 1218.25.

Cy(QLHGP)(SEQ ID NO:17)-PEG₃-biotin. MALDI-MS (m/z): calcd. forC₅₅H₈₉N₁₇O₁₃S (M+H) 1228.65; found 1228.84.

Cy(TLHGP)(SEQ ID NO:18)-PEG₃-biotin. MALDI-MS (m/z): calcd. forC₅₄H₈₈N₁₆O₁₃S (M+H) 1201.64; found 1201.73.

Cy(YDLQR)(SEQ ID NO:19)-PEG₃-biotin. MALDI-MS (m/z): calcd. forC₆₁H₉₈N₁₈O₁₆S (M+H) 1371.71; found 1372.16.

Cy(YDLTR)(SEQ ID NO:20)-PEG₃-biotin. MALDI-MS (m/z): calcd. forC₆₀H₉₇N₁₇O₁₆S (M+H) 1344.70; found 1345.13.

Cy(YDKQR)(SEQ ID NO:21)-PEG₃-biotin. MALDI-MS (m/z): calcd. forC₆₁H₉₉N₁₉O₁₆S (M+H) 1386.72; found 1387.00.

Cy(YDKTR)(SEQ ID NO:22)-PEG3-biotin. MALDI-MS (m/z): calcd. forC₆₀H₉₈N₁₈O₁₆S (M+H) 1359.71; found 1359.94.

Biotin-PEG₃-Cy(KKGWP)(SEQ ID NO:23). MALDI-MS (m/z): calcd. forC₅₉H₉₁N₁₇O₁₃S (M+H) 1278.67; found 1278.83.

Biotin-PEG₃-Cy(KLGWP)(SEQ ID NO:24). MALDI-MS (m/z): calcd. forC₅₉H₉₀N₁₆O₁₃S (M+H) 1263.66; found 1263.92.

Biotin-PEG₃-Cy(LKGWP)(SEQ ID NO:25). MALDI-MS (m/z): calcd. forC₅₉H₉₀N₁₆O₁₃S (M+H) 1263.66; found 1263.86.

Biotin-PEG₃-Cy(LLGWP)(SEQ ID NO:26). MALDI-MS (m/z): calcd. forC₅₉H₈₉N₁₅O₁₃S (M+H) 1248.65; found 1248.89.

Biotin-PEG₃-Cy(RSYNL) (SEQ ID NO:27). MALDI-MS (m/z): calcd. forC₅₇H₉₀N₁₈O₁₆S (M+H) 1315.65; found 1315.95.

Biotin-PEG₃-Cy(RSYNK)(SEQ ID NO:28). MALDI-MS (m/z): calcd. forC₅₇H₉₁N₁₉O₁₆S (M+H) 1330.66; found 1331.06.

Example 5. In Vitro Assays with IL-17F Epitope2 Targeted Ligands

Sandwich ELISA. These assays were performed using the same protocol thatwas used to evaluate the IL-17F Epitope1 targeted ligands.

Results are shown in FIG. 4A. The binding affinity ofPEG₃-biotin-modified Cy(QKHGP) (SEQ ID NO:15), Cy(TKHGP) (SEQ ID NO:16),Cy(KKGWP) (SEQ ID NO:23), and Cy(RSYNK) (SEQ ID NO:28) was tested in anELISA format. For these assays, a dilution series of full-lengthHis-tagged IL-17F protein was captured using the macrocyclic peptideligands immobilized on a NeutrAvidin-coated plate. Cy(QKHGP) (SEQ IDNO:15) and Cy(TKHGP) (SEQ ID NO:16) exhibited EC₅₀ values of 64±10 nMand 72±16 nM, respectively, for human IL-17F protein. Cy(KKGWP) (SEQ IDNO:23) and Cy(RSYNK) (SEQ ID NO:28) exhibited EC₅₀ values of 24±3 nM and15±5 nM, respectively, for human IL-17F protein. Interestingly,Cy(RSYNK) (SEQ ID NO:28) exceeds the binding affinity of the similarlyassayed biotinylated monoclonal anti-IL17F.

Point ELISA (IL-17F vs. IL-17A selectivity assay). A black 96-wellNeutrAvidin Coated High Binding Capacity plate (15510, Pierce) wascoated with 2 μM macrocyclic peptide ligand in TBS (pH 7.6) for 2 h atroom temperature. Biotinylated monoclonal anti-IL17F (TA319597, Origene)was coated at 4 μg/mL in TBS as a control. The plate was aspirated andthen washed with TBS (5×) and Wash Buffer (0.05% (v/v) Tween-20 in PBS,1×). Full-length His-tagged IL-17F (ab167911, Abcam) and IL-17A(ab166882, Abcam) proteins were prepared at 100 nM in Wash Buffer andincubated in the designated microwells for 90 min at room temperature.Microwells were aspirated and subsequently washed with Wash Buffer(10×). To detect the bound IL-17F and IL-17A proteins, AlkalinePhosphatase (AP)-conjugated Anti-6×His Tag® antibody [HIS-1] (ab49746,Abcam) was prepared at 1:10,000 dilution and added to the microwells for1 h at room temperature. The plate was aspirated and washed with WashBuffer (5×). AttoPhos® AP Fluorescent Substrate System (S1000, Promega)was employed to develop the microwells. Using an excitation wavelengthof 430 nm, fluorescent emission at 535 nm was recorded by BeckmanCoulter DTX880 photometer.

Results are shown in FIG. 4B. The selectivity of PEG₃-biotin-modifiedCy(QKHGP) (SEQ ID NO:15), Cy(TKHGP) (SEQ ID NO:16), Cy(KKGWP) (SEQ IDNO:23), and Cy(RSYNK) (SEQ ID NO:28) was tested in an ELISA format. Forthese assays, the full-length His-tagged IL-17F and IL-17A proteins werecaptured using the macrocyclic peptide ligands immobilized on aNeutrAvidin-coated plate. Both Cy(KKGWP) (SEQ ID NO:23) and Cy(RSYNK)(SEQ ID NO:28) exhibited 4:1 selectivity for IL-17F at 100 nM. Otherligands, including Cy(QKHGP) (SEQ ID NO:15) and Cy(TKHGP) (SEQ IDNO:16), and biotinylated monoclonal anti-IL17F show even higher (almostabsolute) selectivity for IL-17F. Again, these results confirm theselective nature of the epitope-targeting strategy.

Example 6. Designing a Linker to Covalently Join Two Macrocyclic Ligands

The tertiary structure of the IL-17F protein was subsequently exploitedas a scaffold for developing a biligand PCC agent that exhibits truecooperative binding (K_(D) range of <1 nM). Combinations of twomacrocycles were covalently joined together to create a biligand PCCagent that displays high avidity for the two epitopes.

Important to this process is the design of suitable linker to bridge thedistance between the two epitopes of the protein. The 3-D crystalstructure of IL-17F (PDB ID: 1JPY) was analyzed in PyMOL (DeLanoScientific) to identify the distances between IL-17F Epitope1 and IL-17FEpitope2 (FIG. 5). In IL-17F Epitope1, focus was placed on the sequenceFFQKPES (SEQ ID NO:1) because the macrocycles Cy(RRATS) (SEQ ID NO:9)and Cy(RRAQS) (SEQ ID NO:10) were determined to interact with thisregion preferentially (FIG. 3). The sequence NENQRVS (SEQ ID NO:3)within IL-17F Epitope2 was the assumed binding region for macrocyclesCy(QKHGP) (SEQ ID NO:15), Cy(TKHGP) (SEQ ID NO:16), Cy(KKGWP) (SEQ IDNO:23), and Cy(RSYNK) (SEQ ID NO:28) because of the N-terminal locationof the click handle. Because IL-17F natively exists as a homodimer,distances were measured between the two epitopes within one and acrossboth monomers. In the monomeric IL-17F protein, the sequence FFQKPES(SEQ ID NO:1) (in IL-17F Epitope1) and IL-17F Epitope2 are separated by˜15 Å. Thus, a chemical linker of ˜15 Å would be useful for covalentlyjoining one macrocycle targeted to IL-17F Epitope1 and one macrocycletargeted to IL-17F Epitope2. The resultant cooperative biligands wouldbe useful for detection or treatment of IL-17F and the IL-17A/Fheterodimer. In the homodimeric IL-17F protein, the sequence FFQKPES(SEQ ID NO:1) (in IL-17F Epitope1) from one monomer and IL-17F Epitope2from the other monomer are separated by ˜7 Å. Thus, a chemical linker of˜7 Å would be useful for covalently joining one macrocycle targeted toIL-17F Epitope1 and one macrocycle targeted to IL-17F Epitope2. Theresultant cooperative biligands would be useful for detection ortreatment of IL-17F.

Example 7. Synthesis of Cooperative Biligand Candidates

Cooperative biligand candidates were synthesized with a variable lengthlinker covalently joining one macrocycle from IL-17F Epitope1 with onemacrocycle from IL-17F Epitope2. The linker connecting the twomacrocycles was a single PEGylated amino acid (Fmoc-NH-PEG_(x)PropionicAcid; x=1 to 5) or glycine (Gly). A PEG linker was chosen because it isavailable in various lengths that would bridge the 7-15 Å distancebetween the two epitopes of the protein. PEG also is expected to displayanti-biofouling properties.

Cooperative biligand candidates were first generated from Cy(RRATS) (SEQID NO:9) (targeted to IL-17F Epitope1) and Cy(QKHGP) (SEQ ID NO:15)(targeted to IL-17F Epitope2). Cy(QKHGP) (SEQ ID NO:15) was prioritizedbased on its high selectivity for IL-17F. Structures of the cooperativebiligand candidates Biotin-PEG₃-Cy(RRATS)(SEQ IDNO:9)-PEG_(x)-Cy(QKHGP)(SEQ ID NO:15) are shown in FIG. 6. Cooperativebiligand candidates were also generated from Cy(RRATS) (SEQ ID NO:9)(targeted to IL-17F Epitope1) and Cy(RSYNK) (SEQ ID NO:28) (targeted toIL-17F Epitope2). Cy(RSYNK) (SEQ ID NO:28) was prioritized based on itshigh affinity for IL-17F.

Example 8. Interleukin 17F (IL17F): Use the Crystal Structure and PCCAssay Data to Estimate the Distance Between the Two Ligands, and Selecta Best Linker from that Information

FIG. 10 shows sequence similarity of IL-17F and IL-17A. FIG. 11 showsthe generation of ligands for IL-17F and IL-17A. Biligand PCCs were madefor IL-17F that bound at two distinct epitopes while monoligand PCs weremade for IL-17A. FIG. 12A provides the full structure of IL-17F (PDB:1JPY). Two PCC-targeted epitopes L1 and L2 are shown to the left of thedrawing. These are two epitopes that distinguish IL-17F from IL-17A.Note that these epitopes are not sequence-adjacent, but are proximalwithin the folded protein structure. FIG. 12B is an expanded view of thetargeted region of the protein (from the crystal structure), and thebinding locations and sequences of two epitope targeted PCC macrocyclicligands are drawn. Each of the PCCs exhibit K_(D) values in the range of15-70 nM.

The PCCs Cy(RRATS) (SEQ ID NO:9) (targeted to IL-17F Epitope1) andCy(RSYNK) (SEQ ID NO:28) (targeted to IL-17F Epitope2) were built withchemical handles for further elaboration. Cy(RSYNK) (SEQ ID NO:28) wasprioritized based on its high affinity for IL-17F (K_(D)=15 nM). Thedistance between those chemical handles, when the PCCs are bound toIL-17F, is an estimated 15 Å. This predicts that an optimized linkerconnecting the two PCCs will have a length near 15 Å. In FIG. 12C thatprediction is validated by testing polyethylene glycol (PEG)oligomer-based linkers of varying lengths. When the two PCCs arecovalently linked to each other by using the PEG oligomer of lengthclosest to 15 Å, the resultant biligand Cy(RSYNK)(SEQ IDNO:28)-PEG₃-Cy(RRATS)(SEQ ID NO:9) exhibits a K_(D) value of 250 pMagainst IL-17F. This represents a >100-fold improvement in affinityrelative to the individual PCC macrocyclic ligands. Shorter or longerlinkers yield biligand affinities that, while improved over either ofthe monoligands, are about ˜10-fold below that of the optimal linker.

Structures of the biligand candidates Biotin-PEG₃-Cy(RSYNK)(SEQ IDNO:28)-PEG_(x)-Cy(RRATS)(SEQ ID NO:9) (x=1 to 5) are shown in FIG. 13.

Example 9. Linkage of the Two Ligands Using Best Available Knowledge,Resulting in a Biligand with Improved Affinity Over the IndividualLigands

Biligand binders to IL-17F with linkers that are shorter (PEG₁, PEG₂) orlonger (PEG₄, PEG₅) than 15 Å exhibit K_(D) values of 1-4 nM againstIL-17F. These values represent a 5- to 25-fold improvement in affinityrelative to the individual PCC macrocyclic ligands.

Plasmodium falciparum Histidine Rich Protein-2 (Pf.HRP-2) is anunstructured protein, but has many epitopes that repeat throughout thestructure. A series of PCCs were developed against various Pf.HRP-2epitopes. The sequence map of Pf.HRP-2 is provided in FIG. 14.

The cyclic peptide, Cy(YKYYR) (SEQ ID NO:29) was developed against theAHHAHHAAD (SEQ ID NO:35) epitope. Cy(YKYYR) (SEQ ID NO:29) exhibits aEC₅₀ of 220 nM. Because of the number of epitope repeats, a cooperativebinder was sought by simply linking two Cy(YKYYR) (SEQ ID NO:29) PCCstogether with about a 1.5 nm long linker. The resultant linked biligandexhibited an EC₅₀ of 20 nM (FIG. 15).

The cyclic PCC with variable sequence GWNVDL (SEQ ID NO:30) wasdeveloped against the C-terminal sequence of Pf.HRP-2(AHHATDAHHAAAHHEAATHCL) (SEQ ID NO:36) (EC₅₀=50 nM). A linker betweenthis PCC and cyclic YKYYR (SEQ ID NO:29) (see above; EC₅₀=220 nM) wasdeveloped by screening a 10,000 element library of variable lengthlinker molecules. The resultant biligand exhibited an EC₅₀ of 540 pM,which is a 100-fold improvement over the better of the two ligandcomponents (FIG. 16).

1-62. (canceled)
 63. A method of producing a synthetic capture agentthat specifically binds to a target protein, the method comprising (a)selecting a first ligand that binds to a first epitope on the targetprotein, (b) selecting a second ligand that binds to a second epitope onthe target protein, (c) selecting a linker that has a length that allowsthe linker to bind both the first ligand and the second ligand when boththe first and the second ligands are specifically binding the first andsecond epitopes, respectively, and (d) binding the linker to the firstand second ligands, thereby producing the synthetic capture agent thatspecifically binds to the target protein.
 64. The method of claim 63,wherein selecting the first ligand is accomplished by screening apeptide library against a first synthetic epitope and identifying afirst library peptide that is coupled to the first synthetic epitopeduring the screening, wherein the first synthetic epitope is a peptidecomprising the sequence of the first epitope and a click handle, whereinthe peptides of the first peptide library each comprise a click handle,wherein the click handle of the first synthetic epitope and the clickhandle of the first library peptides are (1) an azide click handle andan acetylene click handle, respectively, or (2) an acetylene clickhandle and an azide click handle, respectively.
 65. The method of claim64, wherein selecting the second ligand is accomplished by screening asecond peptide library against a second synthetic epitope andidentifying a second library peptide that is coupled to the secondsynthetic epitope during the screening, wherein the second syntheticepitope is a peptide comprising the sequence of the second epitope and aclick handle, wherein the peptides of the second peptide library eachcomprise a click handle, wherein the click handle of the secondsynthetic epitope and the click handle of the second library peptidesare (1) an azide click handle and an acetylene click handle,respectively, or (2) an acetylene click handle and an azide clickhandle, respectively.
 66. The method of claim 63, wherein the linker isselected by estimating the linker length by determining to which side ofthe first epitope the first ligand binds, to which side of the secondepitope the second ligand binds, and measuring the distance between thebound sides of the epitopes on a folded structure of the target protein.67. The method of claim 63, wherein the linker is selected by screeninga library of candidate linker molecules linking the first ligand and thesecond ligand for binding to the target protein.
 68. The method of claim63, wherein the linker is selected by testing a candidate capture agentlinked by a candidate linker for binding to the target protein.
 69. Themethod of claim 63, wherein the first epitope and the second epitope arefrom ˜4.4 Å to ˜26.4 Å, from ˜8.8 Å to ˜26.4 Å, from ˜7 Å to −15 Å or−15 Å distant from each other.
 70. The method of claim 63, wherein thelinker is 10-50% longer than the distance between the first and secondepitopes.
 71. The method of claim 63, wherein the linker is within 5-25%of the distance between the first and second epitopes.
 72. The method ofclaim 63, wherein the linker is within 1-10% of the distance between thefirst and second epitopes.
 73. The method of claim 63, wherein thecapture agent has a binding affinity for the target protein greater thaneither of the ligands.
 74. The method of claim 73, wherein the captureagent has a binding affinity that is at least 50% of the bindingaffinity of a full cooperative binder.
 75. The method of claim 73,wherein the capture agent has a binding affinity that is at least 75% ofthe binding affinity of a full cooperative binder.
 76. The method ofclaim 73, wherein the capture agent has a binding affinity that is atleast 90% of the binding affinity of a full cooperative binder.
 77. Themethod of claim 63, wherein the target protein is a synthetic epitope,wherein the synthetic epitope comprises at least a 20 amino acidsequence of a full length protein, wherein at least one amino acid ofthe synthetic epitope comprises an azide or an acetylene group.
 78. Themethod of claim 77, wherein the full length protein is a naturallyoccurring protein.
 79. The method of claim 78, wherein the naturallyoccurring protein is IL-17.
 80. The method of claim 77, wherein thesynthetic epitope comprises at least a 50 amino acid sequence of a fulllength protein.
 81. The method of claim 77, wherein the capture agentbinds the synthetic epitope and the full length protein with a bindingaffinity that is at least 50% of the binding affinity of a fullcooperative binder.
 82. The method of claim 77, wherein the captureagent binds the synthetic epitope and the full length protein with abinding affinity that is at least 75% of the binding affinity of a fullcooperative binder.
 83. The method of claim 77, wherein the captureagent binds the synthetic epitope and the full length protein with abinding affinity that is at least 90% of the binding affinity of a fullcooperative binder.
 84. A synthetic capture agent produced by (a)selecting a first ligand that binds to a first epitope on the targetprotein, (b) selecting a second ligand that binds to a second epitope onthe target protein, (c) selecting a linker that has a length that allowsthe linker to bind both the first ligand and the second ligand when boththe first and the second ligands are specifically binding the first andsecond epitopes, respectively, and (d) binding the linker to the firstand second ligands, thereby producing the synthetic capture agent thatspecifically binds to the target protein.
 85. The capture agent of claim84, wherein selecting the first ligand is accomplished by screening apeptide library against a first synthetic epitope and identifying afirst library peptide that is coupled to the first synthetic epitopeduring the screening, wherein the first synthetic epitope is a peptidecomprising the sequence of the first epitope and a click handle, whereinthe peptides of the first peptide library each comprise a click handle,wherein the click handle of the first synthetic epitope and the clickhandle of the first library peptides are (1) an azide click handle andan acetylene click handle, respectively, or (2) an acetylene clickhandle and an azide click handle, respectively.
 86. The method of claim85, wherein selecting the second ligand is accomplished by screening asecond peptide library against a second synthetic epitope andidentifying a second library peptide that is coupled to the secondsynthetic epitope during the screening, wherein the second syntheticepitope is a peptide comprising the sequence of the second epitope and aclick handle, wherein the peptides of the second peptide library eachcomprise a click handle, wherein the click handle of the secondsynthetic epitope and the click handle of the second library peptidesare (1) an azide click handle and an acetylene click handle,respectively, or (2) an acetylene click handle and an azide clickhandle, respectively.
 87. The method of claim 84, wherein the linker isselected by estimating the linker length by determining to which side ofthe first epitope the first ligand binds, to which side of the secondepitope the second ligand binds, and measuring the distance between thebound sides of the epitopes on a folded structure of the target protein.88. The method of claim 84, wherein the linker is selected by screeninga library of candidate linker molecules linking the first ligand and thesecond ligand for binding to the target protein.
 89. The method of claim84, wherein the linker is selected by testing a candidate capture agentlinked by a candidate linker for binding to the target protein.
 90. Thecapture agent of claim 84, wherein the first epitope comprises the aminoacid sequence FFQKPES (SEQ ID NO:1).
 91. The capture agent of claim 90,wherein the second epitope comprises the amino acid sequence NENQRVS(SEQ ID NO:3).